3&#39;END CAPS FOR RNAi AGENTS FOR USE IN RNA INTERFERENCE

ABSTRACT

The disclosure relates to novel compounds and compositions comprising a RNAi agent comprising a novel compound as a 3′ end cap. The disclosure also relates to processes for making such compositions, and methods and uses of such compositions, e.g., to mediate RNA interference.

FIELD OF THE INVENTION

The disclosure relates to novel compounds and compositions comprising aRNAi agent comprising a novel compound as a 3′ end cap. The disclosurealso relates to processes for making such compositions, and methods ofmaking and uses for such compositions, e.g., to mediate RNAinterference.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a sequence-specific gene silencing mechanism.This process can be induced artificially by introducing into the cell aRNAi agent targeting a particular sequence. Many structures are suitablefor RNAi agents, including but not limited to short interfering RNAs(siRNAs). RNAi agents can have any of a variety of structures, includingdouble-stranded RNA, which can be modified.

RNAi agents are desirable for therapeutic use. However, this use islimited by a short duration of activity, sometimes mediated by thedegradation of these molecules in blood serum. Naked RNAi agents oftenhave a half-life of minutes. Layzer et al. 2004 RNA 10: 766-771; Chounget al. 2006 Biochem. Biophys. Res. Comm. 342: 919-927; Sato et al. 2007J. Control. Rel. 122: 209-216.

There thus exists the need for novel modifications for RNAi agents whichdo not interfere with RNA interference activity, but which increase theactivity, biological half-life in blood serum, and/or duration ofactivity.

RNAi agents with these modifications would be useful in methods oftarget-specific silencing via the RNA interference mechanism.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present disclosure encompasses a compound offormula Ia:

-   -   in which:

-   X is H; OH, wherein the hydroxyl group can optionally be    functionalized as succinate or attached to a solid support; ODMT;    carboxylic acid; the 3′ end of a strand of a RNAi agent; or the 3′    end of a molecule comprising a strand of a RNAi agent, wherein the    3′ end of the strand terminates in a phosphate or modified    internucleoside linker and further comprises in 5′ to 3′ order: a    spacer, and a second phosphate or modified internucleoside linker;    -   Y is CH or N;    -   m is 0 or 1;    -   p is 1, 2 or 3;    -   R₃ is hydrogen, 2-(hydroxy-methyl)-benzyl,        3-(hydroxy-methyl)-benzyl, succinate, or a solid support (e.g.,        beads or resin);        -   wherein the (CH₂)_(m)—O—R₃ moiety is attached to the phenyl            ring at position 3 or 4;    -   R₄ is hydrogen;    -   R₅ is hydrogen; or R₄ and R₅, together with the phenyl rings to        which R₄ and R₅ are attached, form 6H-benzo[c]chromene.

ODMT is DMT (4,4′-dimethoxytrityl) linked via an oxygen atom.

In various embodiments described herein, a solid support includes,without limitation, bead, resins, or a carrier. A number of suitablesolid supports may be employed for immobilization of the compounds.Examples of suitable solid supports include agarose, cellulose, dextran(commercially available as, i.e., Sephadex, Sepharose) carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper,nitrocellulose, ion exchange resins, plastic films,polyaminemethylvinylether maleic acid copolymer, glass beads, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc.

In various embodiment, the compound of formula Ia is selected from Table1A:

TABLE 1A

In one embodiment, the present disclosure encompasses a compound offormula Ib:

-   -   in which:

-   X is H; OH, wherein the hydroxyl group can optionally be    functionalized as succinate or attached to a solid support; ODMT;    carboxylic acid; the 3′ end of a strand of a RNAi agent; or the 3′    end of a molecule comprising a strand of a RNAi agent, wherein the    3′ end of the strand terminates in a phosphate or modified    internucleoside linker and further comprises in 5′ to 3′ order: a    spacer, and a second phosphate or modified internucleoside linker;    -   q is 0, 1 or 2;    -   R₆ is phenyl which is unsubstituted or substituted with a group        selected from benzoxy and 3,4-dihydroxybutyl;    -   R₇ is hydrogen or hydroxy-ethyl, wherein if R₇ is hydroxy-ethyl,        the hydroxyl can be optionally functionalized as succinate or        attached to a solid support;    -   R₈ is hydrogen or methoxy;    -   Y₁ is CH or N; and    -   Y₂ is N or CR₉; wherein R₉ is selected from hydrogen and methyl.

In various embodiments, the compound of Ib is selected from Table 1B:

TABLE 1B

In various embodiments, the disclosure pertains to a compound selectedfrom Table 1C:

TABLE 1C

-   -   in which:    -   X is H; OH, wherein the hydroxyl group can optionally be        functionalized as succinate or attached to a solid support;        ODMT; carboxylic acid; the 3′ end of a strand of a RNAi agent;        or the 3′ end of a molecule comprising a strand of a RNAi agent,        wherein the 3′ end of the strand terminates in a phosphate or        modified internucleoside linker and further comprises in 5′ to        3′ order: a spacer, and a second phosphate or modified        internucleoside linker, and q is selected from 1 and 2.

In one embodiment, the present disclosure encompasses a method forcapping the 3′ end of a strand of an RNAi agent comprising reacting theRNAi agent with a compound selected from Table 1 D:

TABLE 1D

-   -   in which:

-   X is H; OH, wherein the hydroxyl group can optionally be    functionalized as succinate or attached to a solid support; ODMT;    carboxylic acid; the 3′ end of a strand of a RNAi agent; or the 3′    end of a molecule comprising a strand of a RNAi agent, wherein the    3′ end of the strand terminates in a phosphate or modified    internucleoside linker and further comprises in 5′ to 3′ order: a    spacer, and a second phosphate or modified internucleoside linker,    and q is selected from 1 and 2.

In various embodiments, the disclosure pertains to a DMT-ligand,succinate-ligand and/or carboxylate ligand, such as those listed inTable 4. These are useful in producing an RNAi agent comprising a 3′ endcap comprising a compound of formula Ia or Ib, a compound from any Tableherein, or any 3′ end cap disclosed herein.

In various embodiments, the disclosure pertains to a compound of formulaIa, wherein X is selected from H; OH, wherein the hydroxyl group canoptionally be functionalized as succinate or attached to a solidsupport; ODMT; carboxylic acid; the 3′ end of a strand of a RNAi agent;and the 3′ end of a molecule comprising a strand of a RNAi agent,wherein the 3′ end of the strand terminates in a phosphate or modifiedinternucleoside linker and further comprises in 5′ to 3′ order: aspacer, and a second phosphate or modified internucleoside linker; andR₃ is selected from hydrogen, 2-(hydroxy-methyl)-benzyl,3-(hydroxy-methyl)-benzyl and succinate, or is attached to a solidsupport (e.g., beads or resin).

In various embodiments, the disclosure pertains to a compound of formulaIa, wherein X is the 3′ end of a molecule comprising a strand of a RNAiagent, wherein the 3′ end of the strand terminates in a phosphate ormodified internucleoside linker and further comprises in 5′ to 3′ order:a spacer, and a second phosphate or modified internucleoside linker.

In one embodiment, the disclosure encompasses a RNAi agent comprising afirst strand and a second strand, wherein the 3′-terminus of at leastone strand comprises a 3′ end cap, wherein the 3′ end cap is selectedfrom a compound of formula Ia or Ib (wherein X is 3′ end of a strand ofa RNAi agent), a compound from any Table herein, or any 3′ end capdisclosed herein.

In one embodiment, the first and/or second strands of the RNAi agent areno more than about 49 nucleotides long.

In one embodiment, the first and/or second strands of the RNAi agent areno more than about 30 nucleotides long.

In one embodiment, the first and/or second strand are 19 nucleotideslong.

In one embodiment, the first strand is the anti-sense strand and is 19nucleotides long.

In one embodiment, the RNAi agent has 1 or 2 blunt-ends.

In another embodiment, the RNAi agent comprises an overhang on at leastone 5′ end or 3′ end.

In another embodiment, the RNAi agent comprises a 1 to 6 nucleotideoverhang on at least one 5′ end or 3′ end.

In one embodiment, the RNAi agent comprises a spacer.

In one embodiment, the spacer is a ribitol or other type of abasicnucleotide.

In one embodiment, the spacer is a ribitol or other type of abasicnucleotide, 2′-deoxy-ribitol, diribitol, 2′-methoxyethoxy-ribitol(ribitol with 2′-MOE), C3, C4, C5, C6, or 4-methoxybutane-1,3-diol.

In one embodiment, at least one nucleotide of the RNAi agent ismodified.

In one embodiment, said at least one modified nucleotide is selectedfrom among 2′ alkoxyribonucleotide, 2′ alkoxyalkoxy ribonucleotide, or2′-fluoro ribonucleotide. In another embodiment, said at least onemodified nucleotide is selected from 2′-OMe, 2′-MOE and 2′-H. In variousaspects, the nucleotide subunit is chemically modified at the 2′position of the sugar. In one aspect, the 2′ chemical modification isselected from a halo, a C₁₋₁₀ alkyl, a C₁₋₁₀ alkoxy, a halo, and thelike. In specific aspects, the 2′ chemical modification is a C₁₋₁₀alkoxy selected from —OCH₃ (i.e., “OMe”), —OCH₂CH₃ (i.e., “OEt”) or—CH₂OCH₂CH₃ (i.e., methoxyethyl or “MOE”); or is a halo selected from F.

In various embodiments, one or more nucleotides is modified or is DNA oris replaced by a peptide nucleic acid (PNA), locked nucleic acid (LNA),morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid(GNA), arabinose nucleic acid (ANA), 2′-fl uoroarabinose nucleic acid(FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid(HNA), and/or unlocked nucleic acid (UNA); and/or at least onenucleotide comprises a modified internucleoside linker (e.g., wherein atleast one phosphate of a nucleotide is replaced by a modifiedinternucleoside linker), wherein the modified internucleoside linker isselected from phosphorothioate, phosphorodithioate, phosphoramidate,boranophosphonoate, an amide linker, and a compound of formula (I) (asdescribed elsewhere herein).

In one embodiment, the first two base-pairing nucleotides on the 3′ endof the first and/or second strand are modified.

In one embodiment, the first two base-pairing nucleotides on the 3′ endof the first and/or second strand are 2′-MOE.

In one embodiment, the 3′ terminal phosphate of the first and/or secondstrands is replaced by a modified internucleoside linker.

In another embodiment, the first or second strand is a sense strandcomprising an 5′ end cap which reduces the amount of the RNAinterference mediated by the sense strand.

In various embodiments, the sense strand comprises a 5′ end capselected: a nucleotide lacking a 5′ phosphate or 5′-OH; a nucleotidelacking a 5′ phosphate or a 5′-OH and also comprising a 2-OMe or 2′-MOEmodification; 5′-deoxy-2′-O-methyl modification; 5′-OME-dT; ddT; and5′-OTr-dT.

In various embodiments, the disclosure encompasses a RNAi agentcomprising a first strand and a second strand, wherein the 3′-terminusof at least one strand comprises a 3′ end cap, wherein the 3′ end cap isselected from a compound of formula Ia or Ib, a compound from any Tableherein, or any 3′ end cap disclosed herein; wherein optionally eachstrand is a 49-mer or shorter, optionally the first and/or second strandis about 30 nucleotides long or shorter, and/or optionally the firstand/or second strand is 19 nucleotides long; wherein optionally the RNAiagent has 1 or 2 blunt-ends or the RNAi agent comprises an overhang,optionally a 1 to 6 nucleotide overhang on at least one 5′ end or 3′end; wherein the RNAi agent optionally comprises a spacer, whereinoptionally the spacer is a ribitol or other type of abasic nucleotide,2′-deoxy-ribitol, diribitol, 2′-methoxyethoxy-ribitol (ribitol with2′-MOE), C3, C4, C5, C6, or 4-methoxybutane-1,3-diol; wherein optionallyone or both strands are RNA or optionally at least one nucleotide of theRNAi agent is modified, wherein optionally said at least one modifiednucleotide is selected from among 2′ alkoxyribonucleotide, 2′alkoxyalkoxy ribonucleotide, or 2′-fluoro ribonucleotide, and optionallysaid at least one modified nucleotide is selected from 2′-OMe, 2′-MOEand 2′-H; wherein optionally one or more nucleotides is modified or isDNA or is replaced by a peptide nucleic acid (PNA), locked nucleic acid(LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleicacid (GNA), arabinose nucleic acid (ANA), 2′-fl uoroarabinose nucleicacid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleicacid (HNA), and/or unlocked nucleic acid (UNA) (a non-nucleotide,acyclic analog wherein the C2′-C3′ bond is not present); and/or at leastone nucleotide comprises a modified internucleoside linker, wherein themodified internucleoside linker is selected from phosphorothioate,phosphorodithioate, phosphoramidate, boranophosphonoate, an amidelinker, and a compound of formula (I); and wherein optionally the firsttwo base-pairing nucleotides on the 3′ end of the first and/or secondstrand are modified, and optionally the first two base-pairingnucleotides on the 3′ end of the first and/or second strand are 2′-MOE;and wherein optionally the 3′ terminal phosphate of the first and/orsecond strands is replaced by a modified internucleoside linker; andwherein optionally the first or the second strand is a sense strandcomprising an 5′ end cap which reduces the amount of the RNAinterference mediated by the sense strand, wherein optionally the 5′ endcap selected a nucleotide lacking a 5′ phosphate or 5′-OH; a nucleotidelacking a 5′ phosphate or a 5′-OH and also comprising a 2-OMe or 2′-MOEmodification; 5′-deoxy-2′-O-methyl modification; 5′-OME-dT; ddT; and5′-OTr-dT.

Various elements of various embodiments disclosed herein [e.g.,compositions and methods; and selection of 3′ end caps, nucleotidemodifications or replacements (such as with DNA), patterns ofmodifications, strand length, presence or absence of overhangs, and/or5′ end caps and delivery vehicles] which are not mutually exclusive canbe combined.

In one embodiment, the invention provides a pharmaceutical compositioncomprising an RNAi agent with any one or more of the above properties.

In another embodiment, the invention provides an RNAi agent with any oneor more of the above properties for use as a medicament.

In another embodiment, the disclosure pertains to a method for theinhibition or a method for inhibiting or reducing the level and/oractivity of a target gene in a cell comprising the step of introducinginto the cell one or more of any RNAi agent as described above.

Multiple RNAi agents (which can comprise the same or different types ofRNAi agents, and/or combinations of 3′ end caps, sequences, lengths,overhangs, 5′ end caps, nucleotide replacements and/or modificationsand/or patterns of modification, etc.) can be administered separately orco-administered. The multiple RNAi agents can be administered in thesame delivery vehicle, the same type of delivery vehicle, or indifferent delivery vehicles.

Various additional embodiments are described below.

The details of one or more aspects of the present disclosure are setforth in the accompanying drawings and the description below. Elementsof the various aspects (e.g., sequences, modifications, substitutions,spacers, modified internucleoside linkers, endcaps, combinations of RNAiagents, delivery vehicles, combination therapy involving a RNAi agentand another agent, etc.) disclosed herein or known in the art which arenot mutually exclusive can be combined with each other, provided thatthe agent or agents are still capable of mediating RNA interference. Forexample, any RNAi agent sequence disclosed herein can be combined withany set of modifications or endcaps disclosed herein. Similarly, anycombination of modifications, 5′ end caps, and/or 3′ end caps can beused with any RNAi agent sequence disclosed herein. Any RNAi agentdisclosed herein (with any combination of modifications or endcaps orwithout either modifications or endcaps) can be combined with any otherRNAi agent or other treatment composition or method disclosed herein.

Other features, objects, and advantages of the present disclosure willbe apparent from this description, the drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the structures and sequence of the RNAi agentscomprising a 3′ end cap used in Example 1. The sequences in FIG. 1 arerepresented, from top to bottom, by SEQ ID NO: 1 and 2 (genericsequence); and 3 and 4 (antisense and sense F7). The structures of the3′ end caps (“X”) are provided herein and/or in U.S. Pat. No. 8,084,600.

FIG. 2 shows the efficacy of RNAi agent comprising a 3′ end cap (C3, C6,C12, glycol, cyclohexyl, phenyl, biphenyl, lithochol, C7 amino or C3amino) as described in Example 1. in allowing the RNAi agent to mediateRNA interference. The structures of the 3′ end caps (“X”) are providedherein and/or in U.S. Pat. No. 8,084,600.

FIG. 3 shows the efficacy of the 3′ end caps described in Example 1 inreducing and/or preventing nuclease degradation in serum.

FIG. 4 shows the quality control of various RNAi agents used in Example1.

FIGS. 5A and 5B show residual expression level, indicating in vitro RNAinterference or KD (knockdown) mediated by various RNAi agentscomprising a 3′ end cap: BP (biphenyl), C6, X027, X038, X050, X051,X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067, X068,and X069 on the guide strand, as described in Example 3A. These RNAiagents are without a 2′-MOE clamp (−) or with a 2′-MOE clamp (MOE); orwithout a ribitol spacer (−) or with a ribitol spacer (rib).Descriptions for FIG. 5A are provided at the bottom of FIG. 5B, and thisdata pertains to Example 3A. These are RNAi agents to Hepcidin.

FIGS. 6A and 6B detail some of the RNAi agents used in the data shown inFIGS. 5A and 5B and Example 3A and others. The sequences in FIG. 6A arerepresented by, from top to bottom, by SEQ ID NOs: 5 to 10 (400), 11 to16 (402) and 17 (400 21-mer). The sequences in FIG. 6B are represented,from top to bottom, by SEQ ID NOs: 18 to 23 (400); 24 to 29 (402) and 30(400 21-mer). These are RNAi agents to Hepcidin.

FIG. 7 shows the residual gene activity [wherein residual geneactivity=100%-knock down (KD)] of RNAi agents comprising a 3′ end cap(C6, BP, X027, X058, X067, X038, X069, or X052), at a range from 1.57 nMto 15 nM. The format of the strands is indicated, as described inExample 3A. These are RNAi agents to mouse Hepcidin.

FIGS. 8A and 8B show that in both the ABI Hamp1 Taqman assay (FIG. 8A)and the Hamp1 specific Taqman Assay (FIG. 8B) all of the RNAi agentswith a 3′ end cap were able to mediate knockdown in vivo at 48 hourspost-dose, with a 1×3 mg/kg dose. 3′ end caps used were: X052, X058,X067, X038, X069, and X027, with C6 as a control, as described inExample 3B. These are RNAi agents to mouse Hepcidin tested in vivo.

FIG. 9A shows that the duplex comprising the X058 3′ end cap was stillable to mediate RNA interference (measured by Hepcidin knockdown) at 168hours (7 days) post-dose in vivo, with a 1×3 mg/kg dose, as described inExample 3B. FIG. 9B shows the increased association of the duplexcomprising the X058 3′ end cap with Ago2, compared to the association ofthe duplex comprising the C6 3′ end cap. These are RNAi agents to mouseHepcidin tested in vivo.

FIG. 10 shows the in vivo comparison of RNAi agents of A160 & A161formats and various 3′ end caps (C6 or BP) or a ribitol spacer and a 3′end cap (ribC6), as described in Example 3B. These are human HepcidinRNAi agents.

FIG. 11 (TOP) shows a non-limiting example of the 18-mer RNAi agentformat. “L” indicates a non-nucleotidic “ligand”, e.g., any of various3′ end caps which can be used (e.g., PAZ Ligands). This generic sequenceis represented by SEQ ID NOs: 31 and 32. FIG. 11 (MIDDLE) also showsspecific examples of an 18-mer RNAi agent, comprising at the 3′ terminiof various strands, in 5′ to 3′ order, and bound to the 3′ terminalphosphate: a ribitol spacer (rib), a phosphate (p) and a 3′ end cap(X058 or C6). These sequences are represented by SEQ ID NOs: 33 and 34(400) and 35 and 36 (402). Various modifications are also shown(BOTTOM).

FIG. 12 shows a non-limiting example of synthesis of an RNAi agentcomprising a 3′ end cap (X058), using a succinate form.

FIG. 13 shows the structure of the X058, X109, X110, X111, X112 and X1133′ end caps. TF-26-BC53 indicates a RNAi agent, and these 3′ end capscan be used with either or both strands of any RNAi agent of anysequence or target.

FIG. 14 shows the in vitro efficacy of various RNAi agents comprising a3′ end cap (C6); or, in 5′ to 3′ order, a spacer (C3), a phosphate (p)and a 3′ end cap (C6), or C3pC6. These sequences are represented, fromtop to bottom, by SEQ ID NOs: 37 to 44. The RNAi agents tested are tomouse Factor VII.

FIGS. 15A and 15B shows several different modification schemes for RNAiagents. These RNAi agents comprise a 3′ end cap (C3). FIG. 15B shows amodification scheme in the context of a “wt” (wild-type) and a modifiedRNAi agent, wherein the RNAi agents comprise nucleotidic dTdT or dTsdToverhangs, which can be replaced by a 3′ end cap. FIG. 15C also showsexample modification schemes in the context of a 19 bp or 18 bp stem(double-stranded region), which can further comprise 3′ dinucleotideoverhangs or 3′ end caps (L, or non-nucleotidic Ligands). The variousmodification schemes shown can be used with various RNAi agentscomprising a 3′ end cap as disclosed herein. In FIG. 15A, the genericsequences are represented, from top to bottom, by SEQ ID NOs: 45 to 48.In FIG. 15B, these sequences are represented, from top to bottom, by SEQID NOs: 49 to 52. In FIG. 15C, the generic sequences are represented,from top to bottom, by SEQ ID NOs: 53 to 56. In FIG. 15C, an example19-mer can be converted into a 18-mer by, in one example, deleting theterminal 3′ nucleotide on the antisense strand and the 5′ terminalnucleotide on the sense strand to retain a double-stranded molecule.

FIG. 16A shows the in vitro efficacy of RNAi agents comprising any ofvarious 3′ end caps: X058, X109, X110, X111, X112, X113, or C6 (positivecontrol). FIG. 16B shows the structure of the molecules used in thisexperiment and others. The RNAi agents comprising X109, X110, X111,X112, or X113 comprise a DNA modification at the 5′ end of theanti-sense strand. The duplexes are numbered 20 to 28. The sequences inFIG. 16B are represented by SEQ ID NOs: 57 (first sequence) and 58(second). These are RNAi agents to HuR (ELAVL1). The sequence isdesignated hs (human) 1186, but is cross-reactive between human, mouseand rat.

FIGS. 17A to 17I show data related to 5′ end capping of RNAi agents.FIG. 17A shows two 5′ ends. FIG. 17B shows the effect of 5′ end cappingon HAMP RNAi agents. FIG. 17C diagrams various 5′ end caps. FIG. 17Dillustrates various 5′ end caps and sequences. The sequences in FIG. 17Aare represented, from top to bottom and left to right by SEQ ID NOs: 59to 66. The sequences in FIG. 17C are represented, from top to bottom, bySEQ ID NOs: 67 to 72. The duplexes in FIGS. 17D and 17F are numbered 10to 15.

FIGS. 18A, B and C show the structure and efficacy of various SSB RNAiagents comprising a C6, C8 or C10 3′ end cap, as described in Example 5.FIG. 18C shows example structures of the 3′ end of an RNAi agent strand.The strand terminates in a nucleotide (with BASE) and 3′ phosphate whichis bound to: a dinucleotide (wt or wild-type); or a 3′ end cap (C6, C8or C10). These structures were used in, for example, LNP-formulated SSBsiRNAs, but can be used for any RNAi agent of any length, any sequenceor target. The RNAi agents are to SSB (Sjogren's Syndrome antigen B) andcomprise a 19-mer with a 3′ end cap (C6, C8 or C10). The compounddesignated SSB-309 A22S26 is a 21-mer control. The experiment was donein vivo in mouse. FIG. 18B shows data points used to generate the bargraph shown in FIG. 18A. FIG. 18C also shows that a 3′ terminalphosphate can be replaced by the depicted compound. Any of thephosphates of either or both strands of the RNAi agent can be replacedby the depicted compound.

FIG. 19 diagrams the structure of the 3′ terminal nucleotide of an RNAiagent bound to: a dinucleotide (e.g., CU overhang), or a 3′ end capwhich is a diribitol, ribitol and X027. Also shown are the structures ofa 3′ terminal nucleotide (a 2′-MOE) and phosphate bound to, in 5′ to 3′order: a spacer (ribitol), a second phosphate, and a 3′ end cap (C6 orX058).

FIGS. 20A and B show the efficacy of 3′ end caps and 2′-MOE clampscomprising various modifications. In various RNAi agents, the 3′terminal phosphate (P or PO) of both strands is replaced by aphosphorothioate (P or PS), and the 3′ end cap is a C3. Control RNAiagents lack a 3′ end cap and comprise a 3′ terminal dinucleotide which(dTpdT, where “p” is a phosphate or phosphorothioate). FIGS. 20A and Bshow the efficacy of RNAi agents comprising a 3′ end cap ofphosphorothioate-C3 (PS-C3). FIGS. 20 C, D and E show the efficacy ofRNAi agents comprising a 2′-MOE clamp, wherein the last two base-pairingnt counting from 5′ to 3′ are RNA, DNA, 2′-MOE, 2′-F, or LNA. Thus, invarious RNAi agents, one or more nucleotides is replaced by LNA. For theRNAi agents in FIGS. 20D and 20E, all the tested RNAi agents wereefficacious. It is noted that the percentages do not representknockdown, but knockdown relative to other RNAi agents. 100%, forexample, represents the average knockdown of all antisense strands ofthese efficacious RNAi agents. The sequences in FIG. 20A are representedfrom top to bottom by SEQ ID NOs: 73 to 84. The sequences in FIG. 20Care represented from top to bottom by SEQ ID NOs: 85 and 86.

FIG. 21 shows the structures of example RNAi agents comprising a spacer[which is a ribitol (rib), C3 or 4-methoxybutane-1,3-diol (A5300)]; aphosphate; and a 3′ end cap which is X058. The Figure depicts thespacers in the context of an 18-mer RNAi agent and a specific 3′ endcap, but the spacers can be used with any RNAi agent strand of anylength, sequence or target, and with any 3′ end cap.

FIG. 22 shows the efficacy and duration of RNAi agent activity ofexample RNAi agents comprising a strand, wherein the 3′ end of thestrand terminates in a phosphate and further comprises, in 5′ to 3′order: a spacer which is a ribitol (rib), C3 or 4-methoxybutane-1,3-diol(A5300); a phosphate; and a 3′ end cap which is X058 or C6. The duplexesare numbered 1 to 6. These are RNAi agents to HuR (ELAVL1). UNT:Untreated (negative control). NTC: Non-target control (negative controlusing an unrelated RNAi that targets a different target).

FIG. 23A-C shows efficacy of RNAi agents comprising a 3′ end cap whichis: X109, X110, X111, X112, X113, X1009, X1010, X1024 or X1025 (FIG.23A); X1011, X1012, X1013, X058, X1015, X1016, X1017, X1026, X1027 (FIG.23B): or X1018; X1019, X1020, X1021, X1022 or X1028 (FIG. 23C). Theterms C3 linker, C4 linker, and C5 linker indicate portions of the 3′end cap.

FIGS. 24A and B show efficacy and duration of RNAi agents comprising a3′ end cap which is: X110, X1012, X1018, X111, X1013, X112, X058, X1019,X1025, X1027, or X1028.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure pertains to novel compounds, including: acompound of formula Ia or Ib, a compound from any Table herein, or any3′ end cap disclosed herein.

In various embodiment, the disclosure pertains to a DMT-ligand,succinate-ligand and/or carboxylate ligand, such as those listed inTable 4, which are useful in producing an RNAi agent comprising a 3′ endcap comprising a compound of formula Ia or Ib (e.g., wherein X is H orOH), a compound from any Table herein, or any 3′ end cap disclosedherein.

In various embodiments, the disclosure pertains to a compound of formulaIa, wherein X is selected from H, OH, ODMT, carboxylic acid, and the 3′end of a strand of a RNAi agent; and R₃ is selected from hydrogen,2-(hydroxy-methyl)-benzyl, 3-(hydroxy-methyl)-benzyl and succinate, oris attached to a solid support (e.g, beads or resin).

In various embodiments, the disclosure pertains to a compound designatedherein as X058, wherein X is selected from H, OH, ODMT, carboxylic acid,and the 3′ end of a strand of a RNAi agent; and R₃ is selected fromhydrogen, 2-(hydroxy-methyl)-benzyl, 3-(hydroxy-methyl)-benzyl andsuccinate, or is attached to a solid support (e.g, beads or resin).

In various embodiments, the disclosure pertains to a RNAi agentcomprising a first strand and a second strand, wherein the 3′ end of thefirst and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises a 3′ end cap which is X058.In this case, X represents the first or second strand.

In one embodiment, the compounds of formula Ia and Ib and those of Table1A, 1B or 1C can be used as 3′ end cap on a RNAi agent; in theseembodiments X is the 3′ end of a RNAi agent strand.

In one embodiment, the present disclosure encompasses a RNAi agentcomprising a first strand and a second strand, wherein each strand is a49-mer or shorter, and wherein the 3′-terminus of at least one strandcomprises a 3′ end cap (e.g., a modification at the 3′ end), wherein the3′ end cap is selected from the 3′ end caps listed in Tables 1 or 2 orthose otherwise disclosed herein.

In various embodiments, the present disclosure encompasses a RNAi agentcomprising a sense strand and an antisense strand, wherein each strandis a 49-mer or shorter, and wherein the 3′-terminus of the antisensestrand comprises a 3′ end cap (e.g., a modification at the 3′ end),wherein the 3′ end cap is selected from the 3′ end caps listed in Tables1 or 2 or those otherwise disclosed herein.

In various embodiments, the RNAi agent can be a double-stranded RNA. Invarious embodiments, one or more RNA nucleotides can be replaced by DNA,PNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA. Insome embodiments, the replacement or substitution of RNA with DNA, or anucleotide of a different backbone, or PNA, LNA, Morpholino, TNA, GNA,ANA, HNA, CeNA, FANA can be considered a “modification”. In variousembodiments, one or more phosphates can be replaced by phosphorothioate,phosphorodithioate, phosphoramidate, boranophsophonoate, an amide linkeror a compound of formula (I) (as described elsewhere herein).

In various embodiments, the disclosure pertains to an RNAi agentcomprising a first strand and a second strand, wherein each strand is a49-mer or shorter, and wherein the 3′ end of at least one strandterminates in a phosphate (designated herein as “p” or “PO”) or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a second phosphate or a modified internucleoside linker, and a3′ end cap, wherein the spacer is ribitol, 2′-deoxy-ribitol, diribitol,2′-methoxyethoxy-ribitol (ribitol with 2′-MOE), or a C3, C4, C5 or C6 or4-methoxybutane-1,3-diol; wherein the 3′ end cap is selected from the 3′end caps listed in Tables 1 or 2 or otherwise disclosed herein; whereinoptionally one or more nucleotides is modified or is DNA or is replacedby a peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholinonucleotide, threose nucleic acid (TNA), glycol nucleic acid (TNA),and/or unlocked nucleic acid (UNA); and wherein optionally one or morenucleotides comprise a modified internucleoside linker a modifiedinternucleoside linker (e.g., wherein at least one phosphate of anucleotide is replaced by a modified internucleoside linker).

The 3′ end caps and spacers disclosed herein can be used with one orboth strands of any RNAi agent, regardless of length, sequence ortarget.

Naked siRNAs (e.g., those lacking a 3′ end cap as disclosed) are knownto have a very short biological half-life in blood serum in intestinalfluid, often only minutes. This short half-life may be due todegradation, e.g., by nucleases. Many 3′ end caps have been tested foruse in RNAi agents, though most do not both (1) allow RNA interferenceactivity and (2) increase duration of activity (e.g., reducedegradation). In contrast, 3′ end caps of the present invention are ableto both allow RNA interference and increase time of duration of RNAiagents (e.g., reduce degradation). Preferred 3′ end caps have improvedknockdown (RNA interference activity), and/or further improved durationof activity. Without wishing to be bound by any particular scientifictheory, this disclosure suggests that one or both of these effects mayresult from specific interactions with the PAZ domain of Dicer and/orthrough improvements in stability via reduced exonuclease activity.

In addition, because an RNAi agent is double-stranded, either strand canbe loaded into RISC (the RNA induced silencing complex). The problem isthus that the sense strand can be loaded, but only the antisense strandtargets the correct sequence. The novel 3′ end caps disclosed herein,including those designated “PAZ ligands”, help load the antisensestrand, which increases efficiency, stability and duration of effect.Thus, in some embodiments, the 3′ end of the antisense strand comprisesa 3′ end cap as disclosed herein.

The present disclosure also encompasses methods of decreasing theexpression of a target gene or inhibiting or reducing the level and/oractivity of its gene product, or of treating a disease associated withover-expression of a target gene, in vitro, or in an organism, such as amammal, such as a human being, wherein the method comprises the step ofadministering to the human being a physiologically active amount of acomposition comprising a RNAi agent comprising a first strand and asecond strand, wherein each strand is a 49-mer or shorter, and whereinthe 3′-terminus of at least one strand comprises a 3′ end cap (e.g., amodification at the 3′ carbon), wherein the 3′ end cap is selected fromthe 3′ end caps listed in Tables 1 or 2 or otherwise disclosed herein.

The structures of various 3′ end caps (including those designated “PAZligands”) are shown below in Table 1, below. It is noted that, althoughsome 3′ end caps are designated “PAZ ligands”, this disclosure is notbound by any particular theory.

TABLE 1 STRUCTURES OF 3′ END CAPS (INCLUDING “PAZ LIGANDS”) FOR RNAiAGENTS FOR RNA INTERFERENCE Nickname PAZ ligand C3 Amino

C7 Amino

C3

C6

C8

C10

C12

BP

X027

X038

X050

X051

X052

X058

X059

X060

X061

X062

X063

X064

X065

X066

X067

X068

X069

X097

X098

X109

X110

X111

X112

X113

X1009

X1010

X1011

X1012

X1013

X1015

X1016

X1017

X1018

X1019

X1020

X1021

X1022

X1024

X1025

X1026

X1027

X1028

X1047

X1048

X1049

X1062

X1063

X1064

The structures of Table 1 represent 3′ end caps that can be at the 3′end of one or both strands of an RNAi agent (represented by X). In someembodiments, the 3′ end cap is on the 3′ end of the antisense strand.

Specific embodiments of the structures of Table 1 are diagrammed inTable 2.

In the structures of Tables 1 and 2:

In some embodiments, hydroxyl groups are present and X represents the 3′end of a strand of an RNAi agent. For example, the 3′ end of a strand ofa RNAi can terminate at a phosphate group, to which the 3′ end cap isbound. Non-limiting examples of such a structure are shown in, forexample, FIG. 15A (C3), FIG. 18C (C6, C8, and C10); and FIG. 19 (X027,C6 and X058). Among others, X027 and X058 are active on 19-mers invitro. Table 2 shows the structures of various 3′ end caps bound to thephosphate at the 3′ end of a strand of an RNAi agent. As a non-limitingexample: X058 has been shown (data shown herein and data not shown) tobe a functional 3′ end cap on a variety of different RNAi agents ofdifferent lengths, sequences and targets, both in vitro and in vivo.X058 was an effective 3′ end cap, for example, on 21-mer blunt-ended HuRRNAi agents, wherein each strand is a 21-mer, and the two strandstogether form a blunt-ended duplex, and the 3′ end of each strandterminates in a phosphate and further comprises a 3′ end cap which wasX058. X058 was also effective with several different targets andsequences in the 18-mer format. For example, several effective RNAiagents were constructed comprising a first and a second strand, whereinthe first and second strands both were 18-mers, and the two strandstogether formed a blunt-ended duplex, wherein the 3′ end of the guidestrand terminates in a phosphate or modified internucleoside linker andfurther comprises, in 5′ to 3′ order: a spacer, a phosphate or modifiedinternucleoside linker, and a 3′ end cap which is X058. Severaleffective RNAi agents were constructed comprising a first and a secondstrand, wherein the first and second strands both were 18-mers, and thetwo strands together formed a blunt-ended duplex, wherein the 3′ end ofthe guide strand terminates in a phosphate and further comprises, in 5′to 3′ order: a spacer, a phosphate, and a 3′ end cap which is X058.Several effective RNAi agents were constructed comprising a first and asecond strand, wherein the first and second strands both were 18-mers,and the two strands together formed a blunt-ended duplex, wherein the 3′end of the guide strand terminates in a phosphate and further comprises,in 5′ to 3′ order: a spacer which is ribitol, a phosphate, and a 3′ endcap which is X058. X058 was also effective on other RNAi agents. X058 isthus an effective 3′ end cap on a variety of RNAi agents of differentlengths, targets, and sequences, both in vivo and in vitro.

In some embodiments, where hydroxyl groups are present, the hydroxyl canexist in a protected form. Suitable protecting groups for OH are knownin the art. Protected forms of OH include, but are not limited to,ethers, phosphate esters, methyl tetraacetyl glucuronates, peracetylglycosides and amino acid polypeptide esters.

Embodiments Comprising a Spacer, a Phosphate or Modified InternucleosideLinker, and a 3′ End Cap

In some embodiments, one or both strands of the RNAi agent furthercomprise, at the 3′ end and in 5′ to 3′ order: a spacer, a phosphate ormodified internucleoside linker, and a 3′ end cap (e.g., a 3′ end cap asdisclosed herein).

Thus:

In one embodiment, X is the 3′ end of a molecule comprising a strand ofa RNAi agent, wherein the 3′ end of the strand terminates in a phosphateor modified internucleoside linker and further comprises in 5′ to 3′order: a spacer, and a second phosphate or modified internucleosidelinker. In various embodiments, the spacer is a ribitol,2′-deoxyribitol, or 2′-methoxyethoxy ribitol (ribitol with 2′-MOE), aC3, C4, C5 or C6, or 4-methoxybutane-1,3-diol. Various embodiments aredescribed in more detail below.

SPACERS: Ribitol, Diribitol, 2′-Deoxyribitol, 2′-Methoxyethoxy Ribitol,C3, C4, C5, C6, or 4-Methoxybutane-1,3-Diol (5300)

In some embodiments, the 3′ end of one or both strands of the RNAi agentterminates in a phosphate or modified internucleoside linker and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate or modifiedinternucleoside linker, and a 3′ end cap (e.g., a 3′ end cap asdisclosed herein). A spacer is a chemical moiety intended or used tocreate or maintain a space (e.g., a proper or functional spacing)between two other chemical moieties; e.g., between two phosphates ormodified internucleoside linkers. The spacer can be selected from, forexample, ribitol, diribitol, 2′-deoxyribitol, or 2′-methoxyethoxyribitol (ribitol with 2′-MOE) or an equivalent abasic nucleotide knownto one skilled in the art, or a lower alkyl or alkoxy group such as aC3, C4, C5 or C6, or 4-methoxybutane-1,3-diol, as described below.

Ribitol Spacer.

In some embodiments, the spacer is ribitol or other type of abasicnucleotide.

In one embodiment, the RNAi agent comprises a strand, wherein the 3′ endof the strand terminates in a phosphate or modified internucleosidelinker and further comprises, in 5′ to 3′ order: a spacer which isribitol, a second phosphate or modified internucleoside linker, and a 3′end (e.g., any 3′ end cap described herein or known in the art). Inother words: In one embodiment, the RNAi agent comprises, in 5′ to 3′order: a strand comprising a 3′ terminal phosphate or a modifiedinternucleoside linker; a spacer which is ribitol; a phosphate or amodified internucleoside linker; and a 3′ end cap (e.g., any 3′ end capdescribed herein or known in the art). Thus: In one embodiment, the RNAiagent comprises, in 5′ to 3′ order: a strand comprising a 3′ terminalphosphate; a spacer which is ribitol; a phosphate or a modifiedinternucleoside linker; and a 3′ end cap.

The structure of the 3′ terminal phosphate and ribitol spacer is shownhere:

In some documents, the ribitol spacer is designated as N027 (C0027,etc.).

One embodiment is shown in FIG. 18, wherein the RNAi agent comprises, in5′ to 3′ order: an 18-mer strand, wherein the 3′ end of the 18-merstrand terminates in a phosphate and further comprises, in 5′ to 3′order: a spacer which is ribitol, a phosphate, and a 3′ end cap which isX058. This structure can be on any RNAi strand of any sequence ortarget. In addition, any 3′ end cap disclosed herein can be used inplace of X058.

A related structure is shown in FIG. 19 (“ribitol with X058”), whereinthe last nucleotide of the 18-mer strand is shown (and is a 2′-MOE), andthe 3′ end of the 18-mer strand terminates in a phosphate and furthercomprises, in 5′ to 3′ order: a spacer which is ribitol, a secondphosphate, and a 3′ end cap which is X058.

Another embodiment is shown in FIG. 19 (“ribitol with C6 cap”), whereinthe last nucleotide of the 18-mer strand is shown (and is a 2′-MOE), andthe 3′ end of the 18-mer strand terminates in a phosphate and furthercomprises, in 5′ to 3′ order: a spacer which is ribitol, a phosphate,and a 3′ end cap which is C6.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: an18-mer strand terminating in a 3′ phosphate, a ribitol spacer, aphosphate, and a C6 3′ end cap. This is diagrammed as ribpC6 (or ribC6)in Table 2.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: an18-mer strand terminating in a 3′ phosphate, a ribitol spacer, aphosphate, and a BP 3′ end cap. This is diagrammed as ribpBP (or ribBP)in Table 2.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: an18-mer strand terminating in a 3′ phosphate, a ribitol spacer, aphosphate, and a C10 3′ end cap. This is diagrammed as ribpC10 (orribC10) in Table 2.

One embodiment is shown in FIG. 21, wherein the RNAi agent comprises astrand, wherein the 3′ end of the strand terminates in a phosphate andfurther comprises, in 5′ to 3′ order: a spacer which is ribitol, aphosphate, and a 3′ end cap which is X058. While the structureillustrated in FIG. 21 is an 18-mer RNAi agent, this structure can be onany RNAi strand of any length, sequence or target. In addition, any 3′end cap disclosed herein can be used in place of X058.

In some embodiments, the 3′ end cap is a ribitol. Thus, the RNAi agentcomprises an 18-mer strand, wherein the 3′ end of the 18-mer strandterminates in a phosphate or modified internucleoside linker and furthercomprises, in 5′ to 3′ order: a spacer which is ribitol, a secondphosphate or modified internucleoside linker, and a 3′ end cap which isa second ribitol. In one embodiment, the 3′ end of the 18-mer strandterminates in a phosphate and further comprises, in 5′ to 3′ order: aspacer which is ribitol, a second phosphate, and a 3′ end cap which is asecond ribitol. Such as structure is illustrated in FIG. 19 (includingthe 3′ terminal nucleotide and phosphate) and designated “diribitol”.

In various embodiments, the 3′ end cap is triethylene glycol,cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid),adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050,X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067,X068, X069, X097, X098, X109, X110, X111, X112, X113, X1009, X1010,X1011, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021,X1022, X1024, X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062,X1063, X1064, or ribitol, or any 3′ end cap disclosed herein or known inthe art. The structure comprising an RNAi agent comprising, in 5′ to 3′order, a strand terminating in a 3′ terminal phosphate or a modifiedinternucleoside linker, a ribitol spacer, a phosphate or a modifiedinternucleoside linker, and a 3′ end cap (e.g., any 3′ end cap disclosedherein including but not limited to those listed in the previoussentence) can be used on any RNAi agent of any length, sequence ortarget, including but not limited to a double-stranded RNA, whereinoptionally one or more phosphates are replaced by a modifiedinternucleoside linker, optionally one or more nucleotides are modified,and optionally one or more RNA nucleotides are replaced by DNA, PNA,LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

Diribitol Spacer.

In some embodiments, the spacer is Diribitol.

In one embodiment, the RNAi agent comprises a strand, wherein the 3′ endof the strand terminates in a phosphate or a modified internucleosidelinker and further comprises, in 5′ to 3′ order: a spacer (wherein thespacer comprises in 5′ to 3′ order: a first ribitol; a phosphate or amodified internucleoside linker; a second ribitol; a phosphate or amodified internucleoside linker); and a 3′ end cap.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandcomprising a 3′ terminal phosphate; a first ribitol spacer; a phosphate;a second ribitol spacer; a phosphate or a modified internucleosidelinker; and a 3′ end cap. This structure of the 3′ terminal phosphate,the first ribitol, a phosphate, and the second ribitol is shown here:

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ terminal phosphate or a modified internucleosidelinker, a first ribitol spacer, a phosphate or a modifiedinternucleoside linker, a second ribitol spacer, a phosphate or amodified internucleoside linker, and a 3′ end cap which is a ribitol;this structure is designated a triribitol.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate, a ribitol spacer, a phosphate, and a C63′ end cap. This is diagrammed as ribpC6 (or ribC6) in Table 2.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate, a ribitol spacer, a phosphate, and a BP3′ end cap. This is diagrammed as ribpBP (or ribBP) in Table 2.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate, a ribitol spacer, a phosphate, and a C103′ end cap. This is diagrammed as ribpC10 (or ribC10) in Table 2. Invarious embodiments, the 3′ end cap is triethylene glycol, cyclohexyl,phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052,X058, X059, X060, X061, X062, X063, X064, X065, X066, X067, X068, X069,X097, X098, X109, X110, X111, X112, X113, X1009, X1010, X1011, X1012,X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021, X1022, X1024,X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062, X1063, X1064, orany 3′ end cap disclosed herein or known in the art.

The structure comprising an RNAi agent comprising, in 5′ to 3′ order, astrand terminating in a 3′ terminal phosphate or a modifiedinternucleoside linker, a first ribitol spacer, a phosphate or amodified internucleoside linker, a second ribitol spacer, a phosphate ora modified internucleoside linker, and a 3′ end cap (e.g., any 3′ endcap disclosed herein) can be used on any RNAi agent of any length,sequence or target, including but not limited to a double-stranded RNA,wherein optionally one or more phosphates are replaced by a modifiedinternucleoside linker, optionally one or more nucleotides are modified,and optionally one or more RNA nucleotides are replaced by DNA, PNA,LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

2′-Methoxyethoxy Ribitol Spacer.

In some embodiments, the spacer is 2′-methoxyethoxy ribitol or othertype of abasic nucleotide.

In one embodiment, the RNAi agent comprises a strand, wherein the 3′ endof the strand terminates in a phosphate or modified internucleosidelinker and further comprises, in 5′ to 3′ order: a spacer which is2′-methoxyethoxy ribitol, a second phosphate or modified internucleosidelinker, and a 3′ end (e.g., any 3′ end cap described herein or known inthe art). In other words: In one embodiment, the RNAi agent comprises,in 5′ to 3′ order: a strand comprising a 3′ terminal phosphate or amodified internucleoside linker; a spacer which is 2′-methoxyethoxyribitol; a phosphate or a modified internucleoside linker; and a 3′ endcap (e.g., any 3′ end cap described herein or known in the art). Thus:In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandcomprising a 3′ terminal phosphate; a spacer which is 2′-methoxyethoxyribitol; a phosphate or a modified internucleoside linker; and a 3′ endcap.

The structure of the 3′ terminal phosphate and 2′-methoxyethoxy ribitolspacer is shown here:

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: an18-mer strand, wherein the 3′ end of the 18-mer strand terminates in aphosphate and further comprises, in 5′ to 3′ order: a spacer which is2′-methoxyethoxy ribitol, a phosphate, and a 3′ end cap which is X058.This structure can be on any RNAi strand of any sequence or target. Inaddition, any 3′ end cap disclosed herein can be used in place of X058.

A related structure is 2′-methoxyethoxy ribitol with X058, wherein thelast nucleotide of the 18-mer strand is a 2′-MOE), and the 3′ end of the18-mer strand terminates in a phosphate and further comprises, in 5′ to3′ order: a spacer which is 2′-methoxyethoxy ribitol, a secondphosphate, and a 3′ end cap which is X058.

Another embodiment is 2′-methoxyethoxy ribitol with C6 cap, wherein thelast nucleotide of the 18-mer strand is a 2′-MOE), and the 3′ end of the18-mer strand terminates in a phosphate and further comprises, in 5′ to3′ order: a spacer which is 2′-methoxyethoxy ribitol, a phosphate, and a3′ end cap which is C6.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: an18-mer strand terminating in a 3′ phosphate, a 2′-methoxyethoxy ribitolspacer, a phosphate, and a C6 3′ end cap.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: an18-mer strand terminating in a 3′ phosphate, a 2′-methoxyethoxy ribitolspacer, a phosphate, and a BP 3′ end cap.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: an18-mer strand terminating in a 3′ phosphate, a 2′-methoxyethoxy ribitolspacer, a phosphate, and a C10 3′ end cap.

In another embodiment, the RNAi agent comprises a strand, wherein the 3′end of the strand terminates in a phosphate and further comprises, in 5′to 3′ order: a spacer which is 2′-methoxyethoxy ribitol, a phosphate,and a 3′ end cap which is X058.

In some embodiments, the 3′ end cap is a 2′-methoxyethoxy ribitol. Thus,the RNAi agent comprises an 18-mer strand, wherein the 3′ end of the18-mer strand terminates in a phosphate or modified internucleosidelinker and further comprises, in 5′ to 3′ order: a spacer which is2′-methoxyethoxy ribitol, a second phosphate or modified internucleosidelinker, and a 3′ end cap which is a second 2′-methoxyethoxy ribitol. Inone embodiment, the 3′ end of the 18-mer strand terminates in aphosphate and further comprises, in 5′ to 3′ order: a spacer which is2′-methoxyethoxy ribitol, a second phosphate, and a 3′ end cap which isa second 2′-methoxyethoxy ribitol.

In various embodiments, the 3′ end cap is triethylene glycol,cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid),adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050,X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067,X068, X069, X097, X098, X109, X110, X111, X112, X113, X1009, X1010,X1011, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021,X1022, X1024, X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062,X1063, X1064, or 2′-methoxyethoxy ribitol, or any 3′ end cap disclosedherein or known in the art. The structure comprising an RNAi agentcomprising, in 5′ to 3′ order, a strand terminating in a 3′ terminalphosphate or a modified internucleoside linker, a 2′-methoxyethoxyribitol spacer, a phosphate or a modified internucleoside linker, and a3′ end cap (e.g., any 3′ end cap disclosed herein including but notlimited to those listed in the previous sentence) can be used on anyRNAi agent of any length, sequence or target, including but not limitedto a double-stranded RNA, wherein optionally one or more phosphates arereplaced by a modified internucleoside linker, optionally one or morenucleotides are modified, and optionally one or more RNA nucleotides arereplaced by DNA, PNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA,and/or UNA.

2′-Deoxyribitol Spacer.

In some embodiments the spacer is 2′-deoxyribitol.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate or a modified internucleoside linker, aspacer which is 2′-deoxyribitol (2′-deoxyrib), a phosphate or a modifiedinternucleoside linker, and a 3′ end cap.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate, a spacer which is 2′-deoxyribitol(2′-deoxyrib), a phosphate or a modified internucleoside linker, and a3′ end cap. The structure of the 3′ terminal phosphate and2′-deoxyribitol is shown here:

2′-deoxyribitol (2′-deoxyrib).

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandcomprising a 3′ terminal phosphate; a first ribitol spacer; a phosphate;a second ribitol spacer; a phosphate or a modified internucleosidelinker; and a 3′ end cap.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate, a 2′-deoxyribitol spacer, a phosphate,and a C12 3′ end cap. This is diagrammed as ribpC12 (or ribC12) in Table2. This embodiment is designated “2′DeoxyribC12” and illustrated inTable 2.

In various embodiments, the 3′ end cap is In various embodiments, the 3′end cap is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl),lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6,C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061,X062, X063, X064, X065, X066, X067, X068, X069, X097, X098, X109, X110,X111, X112, X113, X1009, X1010, X1011, X1012, X1013, X1015, X1016,X1017, X1018, X1019, X1020, X1021, X1022, X1024, X1025, X1026, X1027,X1028, X1047, X1048, X1049, X1062, X1063, X1064, or any 3′ end capdisclosed herein or known in the art.

The structure comprising an RNAi agent comprising, in 5′ to 3′ order, astrand terminating in a 3′ terminal phosphate or a modifiedinternucleoside linker, a 2′-deoxyribitol spacer, a phosphate or amodified internucleoside linker, and a 3′ end cap (e.g., any 3′ end capdisclosed herein) can be used on any RNAi agent of any length, sequenceor target, including but not limited to a double-stranded RNA, whereinoptionally one or more phosphates are replaced by a modifiedinternucleoside linker, optionally one or more nucleotides are modified,and optionally one or more RNA nucleotides are replaced by DNA, PNA,LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

C3 Spacer.

In some embodiments the spacer is C3.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate or a modified internucleoside linker, aspacer which is C3, a phosphate or a modified internucleoside linker,and a 3′ end cap.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate, a spacer which is C3, a phosphate or amodified internucleoside linker, and a 3′ end cap.

The C3 spacer has the chemical formula —(CH₂)₃—. The structure of a 3′terminal phosphate and the C3 spacer is shown here:

One embodiment is shown in FIG. 21, wherein the RNAi agent comprises, in5′ to 3′ order: a strand terminating in a 3′ phosphate, a C3 spacer, aphosphate, and a 3′ end cap which is X058. While the structureillustrated in FIG. 21 is an 18-mer RNAi agent, this structure can be onany RNAi strand of any length, sequence or target. In addition, any 3′end cap disclosed herein can be used in place of X058.

Another embodiment is shown in FIG. 14, which illustrates a portion of aRNAi agent to Factor VII comprising a strand, wherein the strandterminates in a phosphate and further comprises in 5′ to 3′ order: a C3spacer, a phosphate and a 3′ end cap which is C6. This is designated“C3pC6 overhang”. This structure can be on any RNAi strand of anysequence or target. In addition, any 3′ end cap disclosed herein orknown in the art can be used in place of C6, and any modifiedinternucleoside linker can be used in place of phosphate.

The efficacy of a RNAi agent comprising a C3 spacer is shown in FIG. 22.Two different HuR constructs were prepared comprising a strand, whereinthe 3′ end of the strand terminates in a phosphate and further comprisesin 5′ to 3′ order: a C3 spacer, a phosphate and a 3′ end cap (which isC6 or X058). Both of these were able to mediate RNA interference.

In various embodiments, the 3′ end cap is triethylene glycol,cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid),adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050,X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067,X068, X069, X097, X098, X109, X110, X111, X112, X113, X1009, X1010,X1011, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021,X1022, X1024, X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062,X1063, X1064, or any 3′ end cap disclosed herein or known in the art.

The structure comprising an RNAi agent comprising, in 5′ to 3′ order, astrand terminating in a 3′ terminal phosphate or a modifiedinternucleoside linker, a C3 spacer, a phosphate or a modifiedinternucleoside linker, and a 3′ end cap (e.g., any 3′ end cap disclosedherein) can be used on any RNAi agent of any length, sequence or target,including but not limited to a double-stranded RNA, wherein optionallyone or more phosphates are replaced by a modified internucleosidelinker, optionally one or more nucleotides are modified, and optionallyone or more RNA nucleotides are replaced by DNA, PNA, LNA, morpholino,TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

C4 Spacer, C5 Spacer and C6 Spacer.

In various embodiments, the spacer is C4 or C5 or C6.

In one embodiment, the RNAi agent comprises a strand, wherein the 3′ endof the strand terminates in a 3′ phosphate or a modified internucleosidelinker, and further comprises a spacer which is C4 or C5 or C6, aphosphate or a modified internucleoside linker, and a 3′ end cap.

In one embodiment, the RNAi agent comprises two strands, wherein the 3′end of each strand terminates in a 3′ phosphate or a modifiedinternucleoside linker, and further comprises a spacer, a secondphosphate or a modified internucleoside linker, and a 3′ end cap,wherein the spacer in one or both strands is C4 or C5 or C6.

The C3 to C6 spacers can be defined as:

C3=1,3-propane-diolC4=1,4-butane-diolC5=1,5-pentane-diolC6=1,6-hexane-diol

In some contexts:

The C4 spacer has the chemical formula —(CH₂)₄—.

The C5 spacer has the chemical formula —(CH₂)₅—.

The C6 spacer has the chemical formula —(CH₂)₆—.

In various embodiments, the 3′ end cap is triethylene glycol,cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid),adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050,X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067,X068, X069, X097, X098, X109, X110, X111, X112, X113, X1009, X1010,X1011, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021,X1022, X1024, X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062,X1063, X1064, or any 3′ end cap disclosed herein or known in the art.

The structure comprising an RNAi agent comprising, in 5′ to 3′ order, astrand terminating in a 3′ terminal phosphate or a modifiedinternucleoside linker, a C4 or C5 or C6 spacer, a phosphate or amodified internucleoside linker, and a 3′ end cap (e.g., any 3′ end capdisclosed herein) can be used on any RNAi agent of any sequence ortarget, including but not limited to a double-stranded RNA, whereinoptionally one or more phosphates are replaced by a modifiedinternucleoside linker, optionally one or more nucleotides are modified,and optionally one or more RNA nucleotides are replaced by DNA, PNA,LNA, morpholino, TNA, GNA and/or UNA.

As a note of clarification, this disclosure notes that the terms “C3”[—(CH₂)₃—], “C4” [—(CH₂)₄—], and “C5” [—(CH₂)₅-] are generally usedherein to designate spacers, similar terms (C3, C4, C5 “linkers”) arealso used to designate a portion of a 3′ end cap. In these figures, thedifferent linkers are used to differentiate portions of various 3′ endcaps. It is also noted that the term “C3” is used to designate a C3 3′end cap (e.g., FIG. 15A), a C3 spacer (FIG. 21), and a C3 linker (FIG.13). The C6 spacer should also be differentiated from the C6 end cap.

4-Methoxybutane-1,3-Diol (5300) Spacer.

In various embodiments, the spacer is 4-methoxybutane-1,3-diol.4-methoxybutane-1,3-diol is also designated 5300, A5300, C5300, G5300,and UG5300.

In one embodiment, the RNAi agent comprises, in 5′ to 3′ order: a strandterminating in a 3′ phosphate (a 3′ terminal phosphate) or a modifiedinternucleoside linker, a spacer which is 4-methoxybutane-1,3-diol, aphosphate or a modified internucleoside linker, and a 3′ end cap.

The structure of a 3′ terminal phosphate and the4-methoxybutane-1,3-diol spacer is shown here:

4-methoxybutane-1,3-diol is also designated 5300, A5300, C5300, G5300,and UG5300. In one embodiment, the RNAi agent comprises, in 5′ to 3′order: a strand terminating in a 3′ phosphate, a spacer which is4-methoxybutane-1,3-diol, a phosphate or a modified internucleosidelinker, and a 3′ end cap.

One embodiment is shown in FIG. 21, wherein the RNAi agent comprises, in5′ to 3′ order: a strand terminating in a 3′ phosphate, a4-methoxybutane-1,3-diol spacer, a phosphate, and a 3′ end cap which isX058. While the structure illustrated in FIG. 21 is an 18-mer RNAiagent, this structure can be on any RNAi strand of any length, sequenceor target. In addition, any 3′ end cap disclosed herein can be used inplace of X058.

The efficacy of a RNAi agent comprising a C5300 spacer is shown in FIG.22. Two different HuR constructs were prepared comprising an 18-mer,wherein the 3′ end of the 18-mer terminates in a phosphate and furthercomprises a C5300 spacer, a phosphate and a 3′ end cap (which is C6 orX058). Both of these were able to mediate RNA interference.

In various embodiments, the 3′ end cap is triethylene glycol,cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid),adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050,X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067,X068, X069, X097, X098, X109, X110, X111, X112, X113, X1009, X1010,X1011, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021,X1022, X1024, X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062,X1063, X1064, or any 3′ end cap disclosed herein or known in the art.

The structure comprising an RNAi agent comprising, in 5′ to 3′ order, astrand terminating in a 3′ terminal phosphate or a modifiedinternucleoside linker, a 4-methoxybutane-1,3-diol spacer, a phosphateor a modified internucleoside linker, and a 3′ end cap (e.g., any 3′ endcap disclosed herein) can be used on any RNAi agent of any length,sequence or target, including but not limited to a double-stranded RNA,wherein optionally one or more phosphates are replaced by a modifiedinternucleoside linker, optionally one or more nucleotides are modified,and optionally one or more RNA nucleotides are replaced by DNA, PNA,LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

Phosphate or Modified Internucleoside Linker

In various embodiments, the modified internucleoside linker is:phosphorothioate, phosphorodithioate, phosphoramidate,boranophosphonoate, an amide linker, or a compound of formula (I), asdetailed below.

In some embodiments, one or both strands of the RNAi agent comprise, atthe 3′ end, a spacer, a phosphate or modified internucleoside linker,and a 3′ end cap (e.g., a 3′ end cap as disclosed herein).

In various embodiments, one or more of the phosphates of one or bothstrands of the RNAi agent are replaced. Thus: In various embodiments,one or more nucleotide of one or both strands has a modifiedinternucleoside linker. In some embodiments, the 3′ terminal phosphateis replaced. In some embodiments, one or more nucleotide of one or bothstrands has a modified internucleoside linker, and/or a modifiedinternucleoside linker is interposed between the spacer and the 3′ endcap.

In one embodiment, the present disclosure encompasses a RNAi agentcomprising a first strand and a second strand, wherein each strand is a49-mer or shorter, and wherein the 3′-terminus of at least one strandcomprises a 3′ end cap, wherein the 3′ end cap is selected from the 3′end caps listed in Tables 1 or 2 or otherwise disclosed herein, andwherein at least one nucleotide has a modified internucleoside linker amodified internucleoside linker (e.g., wherein at least one phosphate ofa nucleotide is replaced by a modified internucleoside linker), wherethe modified internucleoside linker is:

phosphorothioate (PS),

phosphorodithioate, phosphoramidate, boranophosphonoate, an amidelinker, or a compound of formula (I):

where R³ is selected from O—, S—, NH₂, BH₃, CH₃, C₁₋₆ alkyl, C₆₋₁₀ aryl,C₁₋₆ alkoxy and C₆₋₁₀ aryl-oxy, wherein C₁₋₆ alkyl and C₆₋₁₀ aryl areunsubstituted or optionally independently substituted with 1 to 3 groupsindependently selected from halo, hydroxyl and NH₂; and R⁴ is selectedfrom O, S, NH, or CH₂.

FIGS. 20D and E show the efficacy of various RNAi agents wherein the 23′ terminal NT (nucleotides) of the sense (S) or antisense (AS) strandare 2′ MOE phosphate (MOE_PO), 2′OMe phosphate (OMe-PO), RNA (RNA_PO),DNA (DNA_PO), 2′F PS (F_PS), RNA PS (RNA_PS), LNA phosphate (LNA_PO),2′F phosphate (F_PO), 2′OMe PS (OMe_PS), 2′MOE PS (MOE_PS), DNA PS(DNA_PS), or LNA PS (LNA_PS). For the RNAi agents in FIGS. 20D and 20E,all the tested RNAi agents were efficacious. It is noted that thepercentages do not represent knockdown, but knockdown relative to otherRNAi agents. 100%, for example, represents the average knockdown of allantisense strands of these efficacious RNAi agents.

In one embodiment, the present disclosure encompasses a RNAi agentcomprising a first strand and a second strand, wherein each strand is a49-mer or shorter, and wherein the 3′-terminus of at least one strandcomprises a 3′ end cap, wherein the 3′ end cap is selected from the 3′end caps listed in Tables 1 or 2 or otherwise disclosed herein, andwherein at least the 3′ terminal nucleotide on one or both strands has amodified internucleoside linker (e.g., wherein the phosphate of the 3′nucleotide on one or both strands is replaced by a modifiedinternucleoside linker), wherein the modified internucleoside linker isphosphorothioate, phosphorodithioate, phosphoramidate,boranophosphonoate, an amide linker, or a compound of formula (I).

In one embodiment, compounds of table 1 are linked via a terminalphosphate group, which is bound to the 3′ carbon at the 3′ end of atleast one RNAi agent strand. Such compounds are shown in, for example,Table 2.

In one embodiment, compounds of table 2 have a terminal phosphorothioategroup bound to the 3′ carbon at the 3′ end of at least one RNAi agentstrand. Thus, in various embodiments, in the 3′ end caps listed in Table2, the phosphate group is replaced by a phosphorothioate. In additionalembodiments, the phosphate group of various 3′ end caps listed herein asC3, C6, C12, Triethylene glycol, Cyclohexyl, Phenyl, Biphenyl,Adamantane, Lithocholic acid can be replaced by phosphorothioate. In oneparticular embodiment, the phosphate group in the C3 3′ end cap isreplaced by phosphorothioate (and designated “PS—C3”, as illustrated inTable 2 and described in Example 6 and FIGS. 20 A-E). In one particularembodiment, the phosphate group in the C6 3′ end cap is replaced byphosphorothioate (and designated “PS-C6”, as illustrated in Table 2). Inone particular embodiment, the phosphate group in the C10 3′ end cap isreplaced by phosphorothioate (and designated “PS-C10”, as illustrated inTable 2). In one particular embodiment, the phosphate group in thebiphenyl (BP) 3′ end cap is replaced by phosphorothioate (and designated“PS-BP”, as illustrated in Table 2).

In various embodiments, R₁═OH; and R₂=a compound of formula (I). Thisstructure is also shown in FIG. 18C.

3′ End Caps

In some embodiments, one or both strands of the RNAi agent comprise, atthe 3′ end, a spacer, a phosphate or modified internucleoside linker,and a 3′ end cap (e.g., a 3′ end cap as disclosed herein).

A 3′ end cap is a non-nucleotidic chemical moiety bound to the 3′ end ofa molecule comprising a RNAi agent, e.g., the 3′ terminus (or 3′ end) of(a) a molecule comprising a strand, wherein the 3′ end of the strandterminates in a phosphate or modified internucleoside linker; or (b) amolecule comprising, in 5′ to 3′ order: a strand (wherein the 3′ end ofthe strand terminates in a phosphate or modified internucleosidelinker), a spacer, and a second phosphate or modified internucleosidelinker. The 3′ end cap performs at least one of the following functions:allowing RNA interference mediated by the molecule, protecting themolecule from degradation or reducing the amount or rate of degradationof the molecule (e.g., by nucleases), reducing the off-target effects ofthe sense strand, or increasing the activity, duration or efficacy ofRNA interference mediated by the molecule. By describing a 3′ end cap as“non-nucleotidic”, it is meant that a nucleotide comprises threecomponents: a phosphate, a pentose (e.g., a ribose or deoxyribose) and anucleobase, and a 3′ end cap does not comprise all three of thecomponents.

Table 2, below, presents some structures of various 3′ end caps,including some of those shown in Table 1. In several of the structures,the terminal 3′ phosphate of a RNAi agent strand is also shown forcontext, although this phosphate is not part of the 3′ end cap.

Additional information can be found in U.S. patent applications61/886,753; 61/930,681; 61/886,748; 61/886,739; and 61/886,760, whichare all incorporated by reference in their entirety.

TABLE 2 3′ END CAPS FOR RNAi AGENTS FOR USE IN RNA INTERFERENCE 2.A. 3′end caps for use in RNAi agents. Structure (shown bond to phosphate)Nickname (Alternative nickname) In some embodiments, the 3′ end of astrand of a RNAi agent terminates in a phosphate or modifiefinternucleoside linker and optionally further comprises in 5′ to 3′order, a spacer and a phosphate or modified internucleosider linker. Asnon-limiting examples, the phosphate is shown here bound to the 3′ endcap.

C3 amino

C7 amino

C8 amino

C10

X027

X038

X050

X051

X052

X058

X059

X060

X061

X062

X063

X064

X065

X066

X067

X068

X069

X097

X098

X109

X110

X111

X112

X113

X1009

X1011

X1012

X1013

X1015

X1016

X1017

X1018

X1019

X1020

X1021

X1022

X1024

X1025

X1026

X1027

X1028

X1047

X1048

X1049

Ribitol (rib or ribp) 2.B. RNAi agents comprising a strand, wherein the3′ end of the strand terminates in a phosphate or modifiedinternucleoside linker and further comprises in 5′ to 3′ order a spacer,a phosphate or modified internucleoside linker, and a 3′ end cap.Non-limiting examples below show, for example, RNAi agents wherein thespacer is a C3, ribitol or 2′-deoxyribitol. Specific 3′ end caps (C3,C6, C8, C10, C12, BP, C058, etc.) are shown but any 3′ end cap can beused in combination with any spacer or phosphate or modifiedinternucleoside linker.

C3pC6

ribpC3 (ribC3)

ribpC6 (ribC6)

ribpC8 (ribC8)

ribpC10 (ribC10)

ribpC12 (ribC12)

ribpBP (ribBP)

ribpX058 (ribX058)

2′DeoxyribC3

2′DeoxyribC6

2′DeoxyribC8

2′DeoxyribC10

2′DeoxyribC12

2′-DeoxyribBP

Diribitol (dirib or diribp) Additional structures include, inter alia:ribptriethylene glycol, ribpcyclohexyl, ribpphenyl, ribpBP (biphenyl),ribplithochol (lithocholic acid), ribpadamantane, ribpC3 amino, ribpC7amino, ribpC3, ribpC6, ribpC8, ribpC10, ribpC12, ribpX027, ribpX027,ribpX038, ribpX050, ribpX051, ribpX052, ribpX059, ribpX060, ribpX061,ribpX062, ribpX063, ribpX064, ribpX065, ribpX066, ribpX067, ribpX068,ribpX069, ribpX097, ribpX098, ribpX109, ribpX110, ribpX111, ribpX112,ribpX113, ribpX1009, ribpX1011, ribpX1012, ribpX1013, ribpX1015,ribpX1016, ribpX1017, ribpX1018, ribpX1019, ribpX1020, ribpX1021,ribpX1022, ribpX1024, ribpX1025, ribpX1026, ribpX1027, ribpX1028,ribpX1047, ribpX1048, ribpX1049, ribpX1062, ribpX1063, ribpX1064,ribpribitol, etc. These represent a spacer which is ribitol, aphosphate, and a 3′ end cap which is triethylene glycol, cyclohexyl,phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, etc. Additionalstructures include, inter alia: diribptriethylene glycol,diribpcyclohexyl, diribpphenyl, diribpBP (biphenyl), diribplithochol(lithocholic acid), diribpadamantane, diribpC3 amino, diribpC7 amino,diribpC3, diribpC6, diribpC8, diribpC10, diribpC12, diribpX027,diribpX027, diribpX038, diribpX050, diribpX051, diribpX052, diribpX059,diribpX060, diribpX061, diribpX062, diribpX063, diribpX064, diribpX065,diribpX066, diribpX067, diribpX068, diribpX069, diribpX097, diribpX098,diribpX109, diribpX110, diribpX111, diribpX112, diribpX113, diribpX1009,diribpX1011, diribpX1012, diribpX1013, diribpX1015, diribpX1016,diribpX1017, diribpX1018, diribpX1019, diribpX1020, diribpX1021,diribpX1022, diribpX1024, diribpX1025, diribpX1026, diribpX1027,diribpX1028, diribpX1047, diribpX1048, diribpX1049, diribp1062,diribp1063, diribp1064, diribpribitol, etc. These represent a spacerwhich is diribitol, a phosphate, and a 3′ end cap which is triethyleneglycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid),adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050,etc. Additional structures include, inter alia: 2′-deoxyribptriethyleneglycol, 2′-deoxyribpcyclohexyl, 2′-deoxyribpphenyl, 2′-deoxyribpBP(biphenyl), 2′-deoxyribplithochol (lithocholic acid), 2′-deoxyribpadamantane, 2′-deoxyribpC3 amino, 2′-deoxyribpC7 amino,2′-deoxyribpC3, 2′- deoxyribpC6, 2′-deoxyribpC8, 2′-deoxyribpC10,2′-deoxyribpC12, 2′-deoxyribpX027, 2′- deoxyribpX027, 2′-deoxyribpX038,2′-deoxyribpX050, 2′-deoxyribpX051, 2′-deoxyribpX052, 2′- deoxyribpX059,2′-deoxyribpX060, 2′-deoxyribpX061, 2′-deoxyribpX062, 2′-deoxyribpX063,2′- deoxyribpX064, 2′-deoxyribpX065, 2′-deoxyribpX066, 2′-deoxyribpX067,2′-deoxyribpX068, 2′- deoxyribpX069, 2′-deoxyribpX097, 2′-deoxyribpX098,2′-deoxyribpX109, 2′-deoxyribpX110, 2′- deoxyribpX111, 2′-deoxyribpX112,2′-deoxyribpX113, 2′-deoxyribpX1009, 2′-deoxyribpX1011,2′-deoxyribpX1012, 2′-deoxyribpX1013, 2′-deoxyribpX1015,2′-deoxyribpX1016, 2′- deoxyribpX1017, 2′-deoxyribpX1018,2′-deoxyribpX1019, 2′-deoxyribpX1020, 2′- deoxyribpX1021,2′-deoxyribpX1022, 2′-deoxyribpX1024, 2′-deoxyribpX1025, 2′-deoxyribpX1026, 2′-deoxyribpX1027, 2′-deoxyribpX1028, 2′-deoxyribpX1047,2′- deoxyribpX1048, 2′-deoxyribpX1049, 2′-deoxyribp 1062, 2′-deoxyribp1063, 2′-deoxyribp 1064, 2′-deoxyribpribitol, etc. These represent aspacer which is 2′-deoxyribitol, a phosphate, and a 3′ end cap which istriethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol(lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10,C12, X027, X038, X050, etc. Additional structures include, inter alia:C3ptriethylene glycol, C3pcyclohexyl, C3pphenyl, C3pBP (biphenyl),C3plithochol (lithocholic acid), C3padamantane, C3pC3 amino, C3pC7amino, C3pC3, C3pC6, C3pC8, C3pC10, C3pC12, C3pX027, C3pX027, C3pX038,C3pX050, C3pX051, C3pX052, C3pX059, C3pX060, C3pX061, C3pX062, C3pX063,C3pX064, C3pX065, C3pX066, C3pX067, C3pX068, C3pX069, C3pX097, C3pX098,C3pX109, C3pX110, C3pX111, C3pX112, C3pX113, C3pX1009, C3pX1011,C3pX1012, C3pX1013, C3pX1015, C3pX1016, C3pX1017, C3pX1018, C3pX1019,C3pX1020, C3pX1021, C3pX1022, C3pX1024, C3pX1025, C3pX1026, C3pX1027,C3pX1028, C3pX1047, C3pX1048, C3pX1049, C3pX1062, C3pX1063, C3pX1064,C3pribitol, etc. These represent a spacer which is C3, a phosphate, anda 3′ end cap which is triethylene glycol, cyclohexyl, phenyl, BP(biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7amino, C3, C6, C8, C10, C12, X027, X038, X050, etc. Additionalstructures include, inter alia: C4ptriethylene glycol, C4pcyclohexyl,C4pphenyl, C4pBP (biphenyl), C4plithochol (lithocholic acid),C4padamantane, C4pC4 amino, C4pC7 amino, C4pC4, C4pC6, C4pC8, C4pC10,C4pC12, C4pX027, C4pX027, C4pX038, C4pX050, C4pX051, C4pX052, C4pX059,C4pX060, C4pX061, C4pX062, C4pX063, C4pX064, C4pX065, C4pX066, C4pX067,C4pX068, C4pX069, C4pX097, C4pX098, C4pX109, C4pX110, C4pX111, C4pX112,C4pX113, C4pX1009, C4pX1011, C4pX1012, C4pX1013, C4pX1015, C4pX1016,C4pX1017, C4pX1018, C4pX1019, C4pX1020, C4pX1021, C4pX1022, C4pX1024,C4pX1025, C4pX1026, C4pX1027, C4pX1028, C4pX1047, C4pX1048, C4pX1049,C4pX1062, C4p1063, C4p1064, C4pribitol, etc. These represent a spacerwhich is C4, a phosphate, and a 3′ end cap which is triethylene glycol,cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid),adamantane, C4 amino, C7 amino, C4, C6, C8, C10, C12, X027, X038, X050,etc. Additional structures include, inter alia: C5ptriethylene glycol,C5pcyclohexyl, C5pphenyl, C5pBP (biphenyl), C5plithochol (lithocholicacid), C5padamantane, C5pC5 amino, C5pC7 amino, C5pC5, C5pC6, C5pC8,C5pC10, C5pC12, C5pX027, C5pX027, C5pX038, C5pX050, C5pX051, C5pX052,C5pX059, C5pX060, C5pX061, C5pX062, C5pX063, C5pX064, C5pX065, C5pX066,C5pX067, C5pX068, C5pX069, C5pX097, C5pX098, C5pX109, C5pX110, C5pX111,C5pX112, C5pX113, C5pX1009, C5pX1011, C5pX1012, C5pX1013, C5pX1015,C5pX1016, C5pX1017, C5pX1018, C5pX1019, C5pX1020, C5pX1021, C5pX1022,C5pX1024, C5pX1025, C5pX1026, C5pX1027, C5pX1028, C5pX1047, C5pX1048,C5pX1049, C5pX1062, C5pX1063, C5pX1064, C5pribitol, etc. These representa spacer which is C5, a phosphate, and a 3′ end cap which is triethyleneglycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid),adamantane, C5 amino, C7 amino, C5, C6, C8, C10, C12, X027, X038, X050,etc. 2.C. RNAi agents wherein a strand terminates in a 3′ terminalphosphorothioate, and a 3′ end cap. In some RNAi agents of the presentdisclosure, the 3′ end of a strand terminates in a modifiedinternucleoside linker (e.g., a PS) and further comprises a 3′ end cap(C3, C6, etc.). Non- limiting examples of such structures are shownhere, including the 3′ end cap, the terminal modified internucleosidelinker, and, in the first case, sugar and base. In other words, the 3′-terminus of at least one strand comprises a modification at the 3′carbon, wherein the modification is selected from PS-C3, PS-C6, PS-C8,PS-C10, PS-C12, PS-BP, PS-X058, etc., or any modified internucleosidelinker described herein, and any 3′ end cap described herein.

PS-C3

PS-C6

PS-C8

PS-C10

PS-C12

PS-BP

PS-X058

PS-X097

PS-X098

PS-X109

PS-X110

PS-X111 Additional structures include, inter alia: PS-triethyleneglycol, PS-cyclohexyl, PS-phenyl, PS-BP (biphenyl), PS-lithochol(lithocholic acid), PS-adamantane, PS-C3 amino, PS-C7 amino, PS-C3,PS-C6, PS-C8, PS-C10, PS-C12, PS-X027, PS-X027, PS-X038, PS-X050,PS-X051, PS-X052, PS-X059, PS-X060, PS-X061, PS-X062, PS-X063, PS-X064,PS-X065, PS-X066, PS-X067, PS-X068, PS-X069, PS-X097, PS-X098, PS-X109,PS-X110, PS-X111, PS-X112, PS-X113, PS-X1009, PS-X1011, PS-X1012,PS-X1013, PS-X1015, PS-X1016, PS-X1017, PS-X1018, PS-X1019, PS-X1020,PS-X1021, PS-X1022, PS-X1024, PS-X1025, PS-X1026, PS-X1027, PS-X1028,PS-X1047, PS-X1048, PS-X1049, PS-X1062, PS-X1063, PS-X1064, PS-ribitol,etc. These represent phosphorothioate (PS) and a 3′ end cap which istriethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol(lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10,C12, X027, X038, X050, etc.

Regarding Table 2 (including 2.A, 2.B and 2.C):

Synthesis schemes for C7 amino and C3 amino (also designated amino C7 oramino C3, respectively) are not provided, as these molecules arecommercially available from and synthesis schemes were previouslypublished by Glen Research (Sterling, Va.).

C7 amino: Catalog Number: 20-2957-xx; Description: 3′-Amino-Modifier C7CPG 500;2-Dimethoxytrityloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl-longchain alkylamino-CPG; Technical Bulletin: Pre-Synthesis Labeling ofAminomodifier C3 or C7 CPG, Glen Research (Sterling, Va.).

C3 amino: Catalog Number: 20-2913-xx; Description: 3′-Spacer C3 CPG;(1-Dimethoxytrityloxy-propanediol-3-succinoyl)-long chainalkylamino-CPG, Glen Research (Sterling, Va.). Glen Research also notesthat Glen Research has no definitive data on the propyl CPG to supportthe assertion that it protects oligos from exonuclease digestion anddoes not permit polymerase extension. Glen Research's conclusion isbased by analogy to the propylamino-modifier CPG [Zendegui et al.Nucleic Acids Research, 1992, 20, 307-314](Cat. No. 20-2950-41). Thismodification protects oligos from exonuclease digestion but permitspolymerase extension to a small extent since the modifier is eliminatedto a level of about 10% from the 3′ end, leaving the 3′-hydroxyl groupavailable. HPLC experiments have shown that there is no detectableelimination of the propyl group from oligos made from the spacer C3-CPG.

Example 3′ end caps C8 and C10 are also illustrated in FIG. 18C, andribitol and diribitol in FIG. 19, in the context of a RNAi agent strand.

It is noted that Table 2 lists various 3′ end caps that comprise both aspacer (e.g., C3p, ribitol, or 2′-deoxyribitol) and a 3′ end cap. Thus,for example, “C3pC6” can be, depending on context, considered as a “3′endcap”, or as “a spacer and a phosphate and a 3′ end cap” (C3+p+C6) ora spacer, a phosphate and a 3′ end cap. The efficacy of RNAi agentscomprising a spacer and a 3′ end cap is shown in, for example, 5A, 5B,10 and 14.

The present disclosure encompasses any RNAi agent comprising a 3′ endcap as shown in Tables 1 or 2 or otherwise disclosed herein.

Use of 3′ End Caps for Different Sequences and Targets

Various experiments that have shown the 3′ end caps disclosed herein canbe used at the 3′ end of various strands of effective RNAi agents thatcan mediate RNA interference against a variety of different mRNAtargets, including Hepcidin, HuR (ELAVL1), PLK1, SSB and FVII (Factor 7of F7). The 3′ end caps can also be used on RNAi agents for a variety ofspecies, including a variety of mammalian species, as RNAi agentscomprising different 3′ end caps were efficacious in both mouse andhuman cells. Successful RNAi agents comprising various 3′ end capsdisclosed herein were also constructed for several additional genetargets (not described herein). The 3′ end caps described herein havebeen found to be useful in RNAi agents in vivo and in vitro.Furthermore, a variety of successful RNAi agents were constructed andtested wherein one or both strands comprised at the 3′ end, in 5′ to 3′order, a spacer as disclosed herein, a phosphate or internucleosidelinker as disclosed herein, and a 3′ end cap as disclosed herein.

Clearly, as would be known to one of ordinary skill in the art, notevery tested sequence will yield a successful RNAi agent, and certainlynot in combination with any 3′ end cap, or with any spacer, phosphate orinternucleoside linker, and 3′ end cap. However, the 3′ end capsdescribed herein can be used to devise and test various RNAi agents,some of which can have activity approximately equal to that of otherformats (e.g., the canonical structure); and some can produce improvedqualities (e.g., increased activity, duration or activity, decreasedoff-target effects, etc.).

The novel 3′ end caps disclosed herein, therefore, can be used with avariety of different sequences and gene targets.

Hepcidin RNAi Agents Comprising a 3′ End Cap.

As detailed herein e.g., FIGS. 5A to 9, effective RNAi agents comprisingvarious 3′ end caps disclosed herein were constructed targetingHepcidin.

These constructs are detailed in the Figures and Figure legends. Theseconstructs successfully targeted both mouse and human Hepcidin.

Successful 3′ end caps used in these RNAi agents include BP, C6, X027,X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065,X066, X067, X068, X069, etc. Some of these RNAi agents comprise a strandwherein the 3′ end of the strand terminates in a phosphate and furthercomprises a 3′ end cap. Other RNAi agents comprise a strand wherein the3′ end of the strand terminates in a phosphate and further comprises in5′ to 3′ order: a spacer (e.g., ribitol), a phosphate, and a 3′ end cap.

HuR (ELAV1) RNAi Agents Comprising a 3′ End Cap.

Effective RNAi agents to HuR were constructed comprising various 3′ endcaps. See FIGS. 16A and B, and 22-24, Figure legends, etc.

For example: An effective 18-mer RNAi agent to another target, HuR, isshown below:

AS: u U a A u U a U c U a U u C c G u A rib C6 S:C6 rib A a U u A a U a G a U a A g G c A u

n (a, u, c, g): 2′Ome-n (a, u, c, g)

The sequence of the AS (anti-sense) strand, shown above 5′ to 3′, is SEQID NO: 87; the sequence of the S (sense) strand, shown above, 3′ to 5′,is SEQ ID NO: 88. This RNAi agent comprises two strands, eachcomprising, in 5′ to 3′ order, a RNAi agent strand, a spacer (ribitol orrib), a phosphate (not shown), and a 3′ end cap (C6). Other spacers and3′ end caps can be used, and this and other phosphates can be replacedby a modified internucleoside linker.

Other effective HuR RNAi agents were produced wherein the sequence usedwas:

(SEQ ID NO: 89) U002pUpApApU004pU004pApU004pCpU004pApU004pU004pCpCpGpU005pA005pC027pXnnnn Where C0027 is ribitol (or other spacer such as C3 or C5300 as needed).

002=DNA 004=2′Ome 005=2′MOE

All other positions are RNA027=ribitolp=phosphate

Xnnn=3′ end cap (X058, X109, etc.)

In this and various other sequences disclosed herein, U004 indicates anucleotide with a U base with a 2′Ome modification; U002 indicates anucleotide with a U base which is DNA; U005 indicates a nt with a U basewith a 2′MOE modification. Similarly, other nucleotides are modified,e.g., U004 indicates a nucleotide with a U base and a 2′Omemodification.

With this HuR sequence, effective RNAi agents were produced whichcomprise an RNAi agent strand, further comprising at the 3′ end, in 5′to 3′ order: a spacer (ribitol), a phosphate and a 3′ end cap (X058,X109, X110, X111, X112, X113, X1009, X1010, X1011, X1012, X1013, X1015,X1016, X1017, X1018, X1019, X1020, X1021, X1022, X1024, X1025, X1026,X1027, or X1028). These were tested in vitro in Huh-7 cells and alldemonstrated at least about 60% to 80% gene knockdown at 30 pM.

Several of these HuR constructs were further tested, including thosecomprising the RNAi agent strand, further comprising at the 3′ end, in5′ to 3′ order: a spacer (ribitol), a phosphate and a 3′ end cap (X058,X110, X111, X112, X1012, X1013, X1018, X1019, X1025, X1027, X1028).These were tested in vitro in Huh-7 cells and all demonstrated at leastabout 80% to 90% gene knockdown at Day 3 at 1 nM.

Additional HuR constructs comprising a 3′ end cap were constructed,which comprised a strand with the 18-mer sequence above, furthercomprising at the 3′ end, in 5′ to 3′ order: (a) a ribitol spacer, aphosphate and X058 3′ end cap; (b) a ribitol spacer, a phosphate and C63′ end cap; (c) a C3 spacer, a phosphate and a X058 3′ end cap; (d) a C3spacer, a phosphate and a C6 3′ end cap; (e) a C5300 spacer, a phosphateand a X058 3′ end cap; and (f) a C5300 spacer, a phosphate and a C6 3′end cap. Each of these constructs was tested in vitro in Huh-7 cells andall demonstrated about 90% gene knockdown at Day 3 at 1 nM.

Additional RNAi agents to HuR were constructed comprising two strands,each an 18-mer, the two strands together forming a blunt-ended duplex,wherein at least one strand terminates at a 3′ phosphorothioate (PS),the strand further comprising at the 3′ end, in 5′ to 3′ order, a spacer(ribitol), a modified internucleoside linker (another phosphorothioate),and a 3′ end cap (C6).

SSB RNAi Agents Comprising a 3′ End Cap.

Effective RNAi agents were also constructed targeting SSB and comprisinga 3′ end cap, or spacer, phosphate or modified internucleoside linkerand 3′ end cap, as disclosed herein. For example, in some RNAi agents tothese targets, one or both strand further comprises at the 3′ end, in 5′to 3′ order, a spacer (e.g., C3), a phosphate and a 3′ end cap (C6).

For example, the human SSB RNAi agent designatedhs_SSB_309_AS_18mer-C3-C6 was effective at mediating RNA interference invitro, and is shown below:

AS: UuAcAUuAAAGUCUGU87-C3pC6 8 = 2′ methoxy ethyl T; 7 = 2′methoxy ethyl G S: cAAcAGAcuuuAAuGu55-C3pC6 5 = 2′ methoxy ethyl A

n: 2′Ome-n

The sequence of the AS (anti-sense) strand, shown above 5′ to 3′, is SEQID NO: 90. The sequence of the S (sense) strand, shown above 5′ to 3′,is SEQ ID NO: 91.8, 7, 5 and 5 are 2′-MOE nucleosides, as defined asabove.

A variety of RNAi agents targeting SSB and comprising a 3′ end cap orspacer, phosphate or modified internucleoside linker and 3′ end cap, asdisclosed herein, were constructed. These have a variety of targetsequences. For example, in various SSB RNAi agents that wereconstructed, one or both 18-mer strand further comprises, at the 3′ end,in 5′ to 3′ order: a spacer (C3 or ribitol), a phosphate, and a 3′ endcap (C6, BP, a second ribitol, or a diribitol). Other RNAi agents can beconstructed targeting SSB.

Factor VII RNAi Agents Comprising a 3′ End Cap.

A variety of RNAi agents targeting Factor VII (F7) and comprising a 3′end cap (C3, C6, C12, glycol, cyclohex, phenyl, biphenyl, lithochol, C7amino and C3 amino) were constructed; the efficacy of these constructsis shown in FIGS. 1 to 3.

A variety of RNAi agents targeting Factor VII (F7) and comprising a 3′end cap or spacer, phosphate or modified internucleoside linker and 3′end cap, as disclosed herein, were also constructed. This includes, as anon-limiting example, a RNAi comprising a strand further comprising atthe 3′ end, in 5′ to 3′ order, a spacer (C3), a phosphate, and a 3′ endcap (C6). Other RNAi agents can be constructed targeting F7.

PLK1 RNAi Agents Comprising a 3′ End Cap.

A variety of PLK1 RNAi agents comprising a 3′ end cap were constructedand tested.

An RNAi agent was constructed to the target PLK1 and comprising a 3′ endcap or spacer, phosphate or modified internucleoside linker and 3′ endcap, as disclosed herein, e.g., comprising a strand further comprisingat the 3′ end, in 5′ to 3′ order, a spacer (C3), a phosphate, and a 3′end cap (C6).

Improved Activity of 3′ End Caps.

As noted above, in several cases, the RNAi agent comprising a 3′ end capor spacer, phosphate or modified internucleoside linker and 3′ end cap,as disclosed herein, has been shown to have increased activity relativeto a corresponding siRNA lacking such a 3′ end cap. Various siRNAscomprising a 3′ end cap or spacer, phosphate or modified internucleosidelinker and 3′ end cap, as disclosed herein, have shown, in differentexperiments, in vitro and in vivo, to have increased RNA interferenceactivity, increased duration of activity, increased resistance tonuclease degradation, and/or increased specificity. See, for example,FIGS. 1 to 3.

For example, several test siRNAs were constructed against the target F7,including a 21-mer (of the canonical structure, with two dinucleotideoverhangs) and a blunt-ended 18-mer, comprising a C3pC6. In the C3pC6molecule, the 3′ end of the anti-sense strand further comprises, in 5′to 3′ order: a spacer which is C3, a phosphate, and a 3′ end cap whichis C6. Both the 21-mer and 18-mer target the same sequence; both havethe same 5′ start. The RNAi agent comprising the C3pC6, however, showeda lower ED50 (0.44 (±0.022) mg/kg) than the 21-mer (0.61 (±0.017)mg/kg).

In addition, when a Hepcidin RNAi agent (with a X058 3′ end cap) wastested in vivo, it was found, after 2 days, to be more potent than acorresponding 21-mer RNAi agents (which has a dinucleotide overhang).For hepcidin, some RNAi agents with a 3′ end cap as disclosed hereinmore potent than corresponding 21-mers (with a dinucleotide overhang)have been developed.

It was also found that a hepcidin 21-mer siRNA was more prone to sensestrand incorporation into RISC than the corresponding 18-mer with a 3′end comprising a spacer, a phosphate or internucleoside linker and a 3′end cap. Thus, in this case, the 21-mer is less specific.

Thus, in various experiments, the RNAi agent comprising a 3′ endcomprising a spacer, a phosphate or internucleoside linker and a 3′ endcap can demonstrate improved activity compared to a corresponding 21-mersiRNA.

The present disclosure also encompasses methods of decreasing theexpression of a target gene or inhibiting or reducing the level and/oractivity of its gene product, or of treating a disease associated withover-expression of a target gene, in vitro, or in an organism, such as amammal, such as a human being, wherein the method comprises the step ofadministering to the human being a physiologically active amount of acomposition comprising a RNAi agent with a 3′ end comprising a spacer, aphosphate or internucleoside linker and a 3′ end cap, as disclosedherein.

RNAi Agents Comprising a 3′ End Cap

In various embodiments, the disclosure encompasses:

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is C7 amino.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is C3 amino.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is 18 nucleotides, and wherein the 3′ end of at least one strandcomprises a 3′ end cap, and wherein the 3′ end cap is C6. The C6 hasbeen demonstrated to be active in vitro and in vivo with the 18-merformat.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is C8.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is C10. TheC10 has demonstrated beneficial duration of siRNAs with the 18-mer and19-mer format.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is C12. TheC12 has been shown to be active on siRNAs in vitro.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X027.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X038.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X050.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X051.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X052.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X058.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X059.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X060.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X061.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X062.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X063.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X064.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X065.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X066.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X067.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X068.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X069.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X097.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X098.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X109.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X110.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X111.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X112.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X113.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1009.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1010.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1011.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1012.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1013.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1015.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1016.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1017.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1018.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1019.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1020.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1021.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1022.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1024.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1025.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1026.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1027.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1028.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1047.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1048.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1049.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1062.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1063.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is X1064.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the 3′ end of at least onestrand comprises a 3′ end cap, and wherein the 3′ end cap is ribitol.

For each and every of the RNAi agents listed in this section, the RNAiagent can be of any length, sequence or target, and can be, as anon-limiting example, a double-stranded RNA, wherein optionally one ormore phosphates are replaced by a modified internucleoside linker,optionally one or more nucleotides are modified, and optionally one ormore RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA,GNA, ANA, HNA, CeNA, FANA, and/or UNA.

RNAi Agents Terminating in a Phosphate or Modified InternucleosideLinker and Further Comprising in 5′ to 3′ Order: A Spacer, a Phosphateor Modified Internucleoside Linker, and a 3′ End Cap

In various embodiments, the disclosure pertains to an RNAi agentcomprising, in 5′ to 3′ order, a strand terminating in a 3′ terminalphosphate or a modified internucleoside linker, a spacer, a phosphate ora modified internucleoside linker, and a 3′ end cap (e.g., any 3′ endcap listed in Tables 1 or 2 or otherwise disclosed herein).

In various embodiments, the disclosure encompasses:

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is a ribitol.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is a 2′-deoxy-ribitol.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is a diribitol.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is a 2′-methoxyethoxy-ribitol.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is a C3.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is a C4.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is a C5.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is a C6.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or amodified internucleoside linker and further comprises, in 5′ to 3′order: a spacer, a phosphate or a modified internucleoside linker, and a3′ end cap, and wherein the spacer is 4-methoxybutane-1,3-diol.

In each and every RNAi agent in this second strand, the 3′ end cap isselected from: is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl),lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6,C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061,X062, X063, X064, X065, X066, X067, X068, X069, X097, X098, X109, X110,X111, X112, X113, X1009, X1010, X1011, X1012, X1013, X1015, X1016,X1017, X1018, X1019, X1020, X1021, X1022, X1024, X1025, X1026, X1027,X1028, X1047, X1048, X1049, X1062, X1063, X1064, or ribitol. Inaddition, for each and every of the RNAi agents listed in this section,the RNAi agent can be of any length, sequence or target, and can be, asa non-limiting example, a double-stranded RNA, wherein optionally one ormore phosphates are replaced by a modified internucleoside linker,optionally one or more nucleotides are modified, and optionally one ormore RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA,GNA, ANA, HNA, CeNA, FANA, and/or UNA.

Additional Embodiments Comprising a Spacer, a Phosphate or ModifiedInternucleoside Linker, and a 3′ End Cap

This disclosure encompasses, inter alia:

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is C3 and the 3′ end cap is C6. This structure isdesignated C3pC6.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is C3. Thisstructure is designated ribC3 or ribpC3.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is C6. Thisstructure is designated ribpC6. The efficacy of a RNAi agent comprisinga ribpC6 is shown in FIG. 5A. An efficacious RNAi agent comprising this3′ end cap is shown in FIG. 11. RibpC6 is also active in vivo on the18-mer format.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is C6. Thisstructure is designated ribC6 or ribpC6.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is C8. Thisstructure is designated ribC8 or ribpC8.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is C10. Thisstructure is designated ribC10 or ribpC10.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is C12. Thisstructure is designated ribC12 or ribpC12.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is BP. Thisstructure is designated ribpBP. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A. This 3′ end cap is active in vitroin the 18-mer format.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X027. Thisstructure is designated ribX027. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X038. Thisstructure is designated ribX038. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X050. Thisstructure is designated ribX050. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X051. Thisstructure is designated ribX051. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X052. Thisstructure is designated ribX052. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X058. Thisstructure is designated ribX058. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A. An efficacious RNAi agentcomprising this 3′ end cap is shown in FIG. 11.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X059. Thisstructure is designated ribX059. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X060. Thisstructure is designated ribX060. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5A.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X061. Thisstructure is designated ribX061. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X062. This isdesignated ribX062. The efficacy of a RNAi agent comprising this 3′ endcap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X063. Thisstructure is designated ribX063. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X064. ribX064.The efficacy of a RNAi agent comprising this 3′ end cap is shown in FIG.5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X065. Thisstructure is designated ribX065. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X066. Thisstructure is designated ribX066. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X067. Thisstructure is designated ribX067. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X068. Thisstructure is designated ribX068. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X069. Thisstructure is designated ribX069. The efficacy of a RNAi agent comprisingthis 3′ end cap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X097.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X098.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X109.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X110.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X111.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X112.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X113.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1009.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1010.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1011.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1012.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1013.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1015.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1016.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1017.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1018.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1019.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1020.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1021.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1022.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1024.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1025.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1026.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1027.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1028.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1047.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1048.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1049.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1062.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1063.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is X1064.

An RNAi agent comprising a first and a second strand, wherein the firstand/or second strand terminates in a 3′ terminal phosphate and furthercomprises, in 5′ to 3′ order: a spacer, a phosphate, and a 3′ end cap,and wherein the spacer is ribitol and the 3′ end cap is ribitol.

For each and every of structure listed in this section, the RNAi agentcan be of any length, sequence or target, and can be, as a non-limitingexample, a double-stranded RNA, wherein optionally one or morephosphates are replaced by a modified internucleoside linker, optionallyone or more nucleotides are modified, and optionally one or more RNAnucleotides are replaced by DNA, PNA, LNA, morpholino, TNA, GNA, ANA,HNA, CeNA, FANA, and/or UNA.

Additional Embodiments Comprising a Spacer, a Phosphate or ModifiedInternucleoside Linker, and a 3′ End Cap

This disclosure encompasses, inter alia:

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is C3.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is C6.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is C8.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is C10.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is C12.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is BP.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X027.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X038.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X050.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X051.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X052.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X058.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X059.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X060.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X061.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X062. This is designated ribX062.The efficacy of a RNAi agent comprising this 3′ end cap is shown in FIG.5B.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X063.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X064. ribX064. The efficacy of aRNAi agent comprising this 3′ end cap is shown in FIG. 5B.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X065.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X066.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X067.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X068.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X069.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X097.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X098.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X109.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X110.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X111.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X112.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X113.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1009.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1010.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1011.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1012.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1013.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1015.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1016.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1017.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1018.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1019.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1020.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1021.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1022.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1024.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1025.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1026.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1027.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1028.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1047.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1048.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1049.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1062.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1063.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is X1064.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is ribitol.

An RNAi agent comprising a first and a second strand, wherein the 3′ endof the first and/or second strand terminates in a phosphate or modifiedinternucleoside linker and further comprises, in 5′ to 3′ order: aspacer, a phosphate or modified internucleoside linker, and a 3′ endcap, and wherein and the 3′ end cap is triethylene glycol, cyclohexyl,phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052,X058, X059, X060, X061, X062, X063, X064, X065, X066, X067, X068, X069,X097, X098, X109, X110, X111, X112, X113, X1009, X1010, X1011, X1012,X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021, X1022, X1024,X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062, X1063, X1064, orribitol.

For each and every of structure listed in this section, the RNAi agentcan be of any length, sequence or target, and can be, as a non-limitingexample, a double-stranded RNA, wherein optionally one or more phosphateor modified internucleoside linkers are replaced by a modifiedinternucleoside linker, optionally one or more nucleotides are modified,and optionally one or more RNA nucleotides are replaced by DNA, PNA,LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

The disclosure also encompasses a RNAi agent comprising a first strandand a second strand, wherein the first and/or second strand terminatesin a PS (phosphorothioate), and further comprises a 3′ end cap. Thedisclosure also a RNAi agent comprising a first strand and a secondstrand, wherein the first and/or second strand terminates in a PS(phosphorothioate), and further comprises, in 5′ to 3′ order: a spacer,phosphate or a modified internucleoside linker, and a 3′ end cap.

Thus, the disclosure encompasses:

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the first and/or secondstrand terminates in a PS and further comprises a 3′ end cap, whereinthe 3′ end cap is C3. This structure is designated PS-C3. This efficacyof a RNAi agent comprising this 3′ end cap is described in Example 6 andFIGS. 20 A-E).

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the first and/or secondstrand terminates in a PS and further comprises a 3′ end cap, whereinthe 3′ end cap is C6. This structure is designated PS-C6.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the first and/or secondstrand terminates in a PS and further comprises a 3′ end cap, whereinthe 3′ end cap is C8. This structure is designated PS-C8.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the first and/or secondstrand terminates in a PS and further comprises a 3′ end cap, whereinthe 3′ end cap is C10. This structure is designated PS-C10.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the first and/or secondstrand terminates in a PS and further comprises a 3′ end cap, whereinthe 3′ end cap is C12. This structure is designated PS-C12.

A RNAi agent comprising a first strand and a second strand, wherein eachstrand is a 49-mer or shorter, and wherein the first and/or secondstrand terminates in a PS and further comprises a 3′ end cap, whereinthe 3′ end cap is BP. This structure is designated PS-BP.

Alternative Names and DMT, Succinate and Carboxylate Variants of 3′ EndCaps

In various embodiments, the disclosure encompasses DMT, succinate andcarboxylate forms of the various 3′ end caps, which can be used toconstruct RNAi agents comprising a 3′ end cap (or a spacer and a 3′ endcap). In the compounds shown in Table 1, for example, independently,R1=OH, succinate or protected forms of OH; and R2=ODMT (where ODMT isDMT (4,4′-dimethoxytrityl) linked via an oxygen atom), or carboxylate.Protected forms of OH include, but are not limited to, ethers, phosphateesters, methyl tetraacetyl glucuronates, peracetyl glycosides and aminoacid polypeptide esters.

Alternative nicknames have been devised for various DMT, Succinate andCarboxylate forms of various 3′ end caps (also called “ligands”).

The succinate form can optionally be used to load these molecules ontosolid support for RNAi agent synthesis. One such synthesis is shown inFIG. 12, although other routes are also possible.

In addition to the succinyl (CO2H—(CH2)n-CO2H; n=2) linker used forsolid-phase φG loading and oligonuceotide synthesis, other diacids ofvarying length [CO2H—(CH2)n-CO2H] can be used, as well as other“universal support” strategies known in the art, (for example, GlenUnySupport™ from Glen Research, Sterling, Va.), includinghydroquinone-O,O′-diacyl (Pon et al. Nucl. Acids Res. 1997, 18,3629-3635),N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6-(4,4′-dimethoxytrityloxy)-5-succinoyl(Guzaev et al. J. Am. Chem. Soc. 2003, 125, 2380-2381; Kumar et al.Tetrahedron 2006, 62, 4528-4534),(2S,3S,4R,5R)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-2,5-dimethoxytetrahydrofuran-3-succinoyl(Scott et al. “Innovations and Perspectives in Solid Phase Synthesis,3rd International Symposium,” 1994, Ed. Roger Epton, MayflowerWorldwide, 115-124), and1-Dimethoxytrityloxy-2-O-dichloroacetyl-propyl-3-N-succinyl (Azhayev.Tetrahedron, 1999, 55, 787-800; Azhayev et al. Tetrahedron 2001, 57,4977-4986).

The succinate is traceless as it is only a synthetic handle not presentin the final form of the siRNA.

It is noted, though, that the PAZ binding assay using the carboxylatevariant was not predictive of efficacy, since several of the disclosedPAZ ligands below did not bind the PAZ ligand in this assay, but werelater found to be effective as 3′ caps when conjugated to an siRNA.

The structures of DMT-ligand, Succinate-ligand and carboxylate forms ofthe 3′ end caps are shown in Table 4, below.

TABLE 4 DMT-LIGAND, SUCCINATE-LIGAND, AND CARBOXYLATE FORMS OF 3′ ENDCAPS Ligand DMT-ligand Succinate-ligand Carboxylate X027

X038

X050

X051

X052

X058

X059

X060

X061

X062

X063

X064

X065

X066

X067

X068

X069

RNAi Agents Comprising a 3′ End Cap

The present disclosure thus provides a double-stranded RNAi agent (adouble-stranded molecule capable of mediating RNA interference,including but not limited to a siRNA) comprising a first and a secondstrand, wherein the first and/or second strand comprise at least 15 toat least 19 or more contiguous nucleotides of target gene, wherein theRNAi agent comprises a 3′ end cap on one or both strands. The first andsecond strand can be, depending on context, an antisense and a sensestrand, or a sense and an antisense strand. The sense and anti-sensestrand can be non-contiguous, contiguous, or covalently bound, e.g., viaa loop or linker. In particular, the 3′ end cap is selected from thoselisted in Tables 1 or 2 or otherwise disclosed herein. If both strandscomprise a 3′ end cap, the 3′ end cap on each strand can be the same ordifferent. The RNAi agent particularly can in one embodiment compriseless than 30 nucleotides per strand, e.g., such as 17-23 nucleotides,15-19, 18-22 nucleotides, and/or 19-21 nucleotides, and be modified andunmodified (e.g., at the 2′ carbon) at one or more nucleotides.

The double-stranded RNAi agents can have 0, 1 or 2 blunt ends, and/oroverhangs [e.g., of 1, 2, 3 or 4 nucleotides (i.e., 1 to 4 nt)] from oneor both 3′ and/or 5′ ends.

The RNAi agent can either contain only naturally-occurring nucleotidesubunits (e.g., ribonucleotides), or one or more modifications to thesugar, phosphate or base of one or more of the replacement nucleotidesubunits, whether they comprise ribonucleotide subunits ordeoxyribonucleotide subunits or other related modified variants. RNAiagents thus include those that contain substitutions of anaturally-occurring nucleotide by an alternative backbone nucleotide(e.g., a PNA, morpholino, LNA, TNA, GNA, ANA, HNA, CeNA, FANA, and/orUNA, etc.) and/or a modified nucleotide.

In one embodiment, modified variants of the disclosed RNAi agents have athymidine (as RNA, or, preferably, DNA) replacing a uridine, or have aninosine base. In some embodiments, the modified variants of thedisclosed RNAi agents can have a nick in the “passenger” (aka “sense”)strand, mismatches between the guide and passenger strand, DNA replacingthe RNA of a portion of both the guide and passenger strand (e.g., theseed region), and/or a shortened passenger strand (e.g., 15, 16, 17 or18 nt). Once a functional 3′ end cap suitable for use with a guidestrand is identified, modifications and variants of the RNAi agent canbe readily made. The disclosed 3′ end caps can be used in any RNAi agentcomprising any combination of embodiments or features which are notmutually exclusive (e.g., the combination of base modifications withshortened passenger strand; or nicked passenger strand and basemodifications; or DNA replacing part or all of the seed region and basemodifications in the remaining RNA; or the combination of modificationswith any delivery vehicle; etc.).

In one embodiment, the modifications improve efficacy, stability (e.g.,against nucleases in, for example, blood serum or intestinal fluid),and/or reduce immunogenicity of the RNAi agent. One embodiment of thepresent disclosure relates to a double-stranded oligonucleotidecomprising at least one non-natural nucleobase. In certain embodiments,the non-natural nucleobase is difluorotolyl, nitroindolyl,nitropyrrolyl, or nitroimidazolyl. In a particular embodiment, thenon-natural nucleobase is difluorotolyl. In certain embodiments, onlyone of the two oligonucleotide strands contains a non-naturalnucleobase. In certain embodiments, both of the oligonucleotide strandscontain a non-natural nucleobase.

The RNAi agent(s) can optionally be attached to a ligand selected toimprove one or more characteristic, such as, e.g., stability,distribution and/or cellular uptake of the agent, e.g., cholesterol or aderivative thereof. The RNAi agent(s) can be isolated or be part of apharmaceutical composition used for the methods described herein.Particularly, the pharmaceutical composition can be formulated fordelivery to specific tissues (e.g., those afflicted with a targetgene-related disease) or formulated for parenteral administration. Thepharmaceutical composition can optionally comprise two or more types orsequences of RNAi agents, each one directed to the same or a differentsegment of the target gene mRNA. Optionally, the pharmaceuticalcomposition can further comprise or be used in conjunction with anyknown treatment for any target gene-related disease. The pharmaceuticalcomposition can comprise a RNAi agent comprising a 3′ end cap and anysuitable delivery vehicle disclosed herein or known in the art.

The present disclosure further provides methods for inhibiting orreducing the level and/or activity of target gene mRNA in a cell,particularly in the case of a disease characterized by over-expressionor hyper-activity of the target gene. Cells comprising an alterationsuch as a mutation, over-expression and/or hyperactivity of target geneare termed “target gene-defective” cells. Such methods comprise the stepof administering one or more of the RNAi agents of the presentdisclosure to a cell, as further described below. The present methodsutilize the cellular mechanisms involved in RNA interference toselectively degrade the target RNA in a cell and are comprised of thestep of contacting a cell with one or more of the RNAi agents of thepresent disclosure.

The present disclosure also encompasses a method of treating a humansubject having a pathological state mediated at least in part by targetgene expression, the method comprising the step of administering to thesubject a therapeutically effective amount of a RNAi agent targeting thetarget gene.

The methods and compositions of the present disclosure, e.g., themethods and target gene RNAi agent compositions, can be used in anyappropriate dosage and/or formulation described herein or known in theart, as well as with any suitable route of administration describedherein or known in the art.

The method also optionally further comprises the step of administering asecond agent. In some embodiments, this second agent is another RNAiagent to target gene. In other embodiments, the second agent is anothertreatment, such as one directed to another target, which is alsohyper-active, mutated and/or over-expressed in the pathological state.

The details of one or more embodiments of the present disclosure are setforth in the accompanying drawings and the description below. Elementsof the various embodiments (e.g., 3′ end caps, sequences, modifications,patterns of modifications, 5′ end caps, combinations of RNAi agents,combination therapy involving a target gene RNAi agent and anotheragent, etc.) which are not mutually-exclusive can be combined with eachother as described herein and as known or developed in the art. Forexample, any 3′ end cap disclosed herein can be combined with any set ofmodifications or sequence disclosed herein. Any combination ofmodifications, 5′ end caps, and/or sequence can be used with any 3′ endcap disclosed herein. Any RNAi agent disclosed herein (with anycombination of modifications or endcaps or without either modificationsor endcaps) can be combined with any other RNAi agent or other treatmentcomposition or method disclosed herein.

Thus, the present disclosure encompasses any RNAi agent disclosedherein, or any method involving any RNAi agent disclosed herein, whereinthe RNAi agent comprises at least one 3′ end cap as disclosed herein.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“Alkyl” is a monovalent saturated hydrocarbon chain having the specifiednumber of carbon atoms. For example, C₁₋₆ alkyl refers to an alkyl grouphaving from 1 to 6 carbon atoms. Alkyl groups may be straight orbranched. Representative branched alkyl groups have one, two, or threebranches. Examples of alkyl groups include, but are not limited to,methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl,isobutyl, sec-butyl, and t-butyl), pentyl (n-pentyl, isopentyl, andneopentyl), and hexyl.

“Aryl” is a hydrocarbon ring system having an aromatic ring. Aryl groupsare monocyclic ring systems or bicyclic ring systems. Monocyclic arylring refers to phenyl. Bicyclic aryl rings refer to naphthyl and torings wherein phenyl is fused to a C₅₋₇ cycloalkyl or C₅₋₇ cycloalkenylring as defined herein.

RNA Interference

As used herein, “RNA interference” (RNAi) is a post-transcriptional,targeted gene-silencing technique that uses a RNAi agent to degrademessenger RNA (mRNA) containing a sequence which is the same as or verysimilar to the RNAi agent. See: Zamore and Haley, 2005, Science, 309,1519-1524; Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001,Nature, 411, 494-498; and Kreutzer et al., PCT Publication WO 00/44895;Fire, PCT Publication WO 99/32619; Mello and Fire, PCT Publication WO01/29058; and the like. The process of RNAi occurs naturally when longdsRNA is introduced into a cell and cleaved by ribonuclease III (Dicer)into shorter fragments called siRNAs. Naturally produced siRNAs aretypically about 21 nucleotides long and comprise about 19 base pairduplexes with two 2-nt overhangs (the “canonical” structure). One strandof the siRNA is incorporated into the RNA-induced silencing complex(RISC). This strand (known as the anti-sense or guide strand strand)guides RISC to a complementary mRNA. One or more nucleases in the RISCthen mediates cleavage of the target mRNA to induce silencing. Cleavageof the target RNA takes place in the middle of the region complementaryto the anti-sense strand. See: Nykanen, et al. 2001 Cell 107:309; Sharpet al. 2001 Genes Dev. 15:485; Bernstein, et al. 2001 Nature 409:363;Elbashir, et al. 2001 Genes Dev. 15:188.

As used herein, the term “RNAi agent” encompasses siRNAs (including butnot limited to those of the “canonical” structure), in addition tovarious natural and artificial structures capable of mediating RNAinterference. As detailed below, these structures can be longer orshorter than the canonical, and/or blunt-ended, and/or comprise one ormore modification, mismatch, gap, and/or nucleotide replacement. The 3′end caps of the present disclosure can be used with any RNAi agent andcan allow two functions: (1) allowing RNA interference; and (2)increasing duration of activity and/or biological half-life, which maybe accomplished, for example, by increased binding to the PAZ domain ofDicer and/or reducing or preventing degradation of the RNAi agent (e.g.,by nucleases such as those in the serum or intestinal fluid).

The RNAi agent(s) of the present disclosure target (e.g., bind to,anneal to, etc.) the target mRNA. The use of the RNAi agent to thetarget results in a decrease of target activity, level and/orexpression, e.g., a “knock-down” or “knock-out” of the target gene ortarget sequence. Particularly, in one embodiment, in the case of adisease state characterized by over-expression or hyper-activity oftarget gene, administration of a RNAi agent to target gene knocks downthe target gene target enough to restore a normal level of target geneactivity.

A suitable RNAi agent can be selected by any process known in the art orconceivable by one of ordinary skill in the art. For example, theselection criteria can include one or more of the following steps:initial analysis of the target gene sequence and design of RNAi agents;this design can take into consideration sequence similarity acrossspecies (human, cynomolgus, mouse, etc.) and dissimilarity to other(non-target gene) genes; screening of RNAi agents in vitro (e.g., at 10nM in cells); determination of EC50 in cells; determination of viabilityof cells treated with RNAi agents, including insensitive cells which donot require target gene for survival, or sensitive cells, which dorequire target gene for survival; testing with human PBMC (peripheralblood mononuclear cells), e.g., to test levels of TNF-alpha to estimateimmunogenicity, wherein immunostimulatory sequences are less desired;testing in human whole blood assay, wherein fresh human blood is treatedwith an RNAi agent and cytokine/chemokine levels are determined [e.g.,TNF-alpha (tumor necrosis factor-alpha) and/or MCP1 (monocytechemotactic protein 1)], wherein Immunostimulatory sequences are lessdesired; determination of gene knockdown in vivo using subcutaneoustumors in test animals; target gene target gene modulation analysis,e.g., using a pharmacodynamic (PD) marker, for example, other factorswhose expression is affected by target gene, wherein target geneknockdown leads to a dose-dependent reduction of abundance of thosecomponents; and optimization of specific modifications of the RNAiagents.

RNAi agents comprising a 3′ end cap described herein are thus useful inRNA interference of target gene.

It is known in the art that naked siRNA (lacking a suitable 3′ end cap,such as those disclosed herein) has a short duration of activity invivo; it is rapidly degraded by nucleases in serum, often with ahalf-life of minutes. Layzer et al. 2004 RNA 10: 766-771; Choung et al.2006 Biochem. Biophys. Res. Comm. 342: 919-927; and Sato et al. 2007 J.Control. Rel. 122: 209-216. Many 3′ end caps previously described do notboth allow RNA interference and either protect the molecules fromnucleases or extend time of duration.

RNAi agents comprising 3′ end caps disclosed herein mediate theseactivities.

Non-limiting examples of RNAi agent structures suitable for use with thedisclosed 3′ end caps are described below.

Structure of a RNAi Agent: Antisense Strand and Sense Strand of VariousLengths, (Optional) Overhangs, (Optional) 5′ End Caps, (Optional)Modifications; (Optional) Patterns of Modification.

RNAi agents mediate RNA interference and comprise a first strand and asecond strand, e.g., a sense strand and an antisense strand (or anantisense and a sense strand), wherein the strands are optionallyprimarily RNA (optionally wherein one or more nucleotides are replacedand/or modified), (optionally) further comprising one or two overhangs,and (optionally) one or two 5′ end caps, wherein the optionalmodifications can optionally be in various patterns of modification, andthe strands can optionally be of varyious lengths. RNAi agents of thepresent disclosure comprise a 3′ end cap on either the sense and/oranti-sense strand.

Anti-Sense and Sense Strands

The term “antisense strand” (AS), as used herein, refers to the strandof a RNAi agent which includes a region that is fully or substantiallycomplementary to a target sequence. The “antisense strand” is sometimestermed the “guide” strand. As used herein, the term “region ofcomplementarity” refers to the region on the antisense strand that isfully or substantially complementary to a target mRNA sequence. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches may be in the internal or terminal regions ofthe molecule. Generally, the most tolerated mismatches are in theterminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′and/or 3′ end. The portion of antisense strand most sensitive tomismatches is termed the “seed region”. In a RNAi agent comprisingstrands of exactly 19 nt, the 19^(th) position (counting from 5′ to 3′)can tolerate some mismatches.

The term “sense strand” (S), as used herein, refers to the strand of aRNAi agent that includes a region that is substantially complementary toa region of the antisense strand as that term is defined herein. The“sense” strand is sometimes termed the “passenger” or “anti-guide”strand. By their sequences, the antisense strand targets the desiredmRNA, while the sense strands targets a different target. Thus, if theantisense strand is incorporated into RISC, the correct target istargeted. Incorporation of the sense strand can lead to off-targeteffects. These off-target effects can be limited by use ofmodifications, or use of 5′ end caps on the sense strand, as describedbelow.

The sequence of a gene may vary from individual to individual,especially at wobble positions within the coding segment, or in theuntranslated region; individuals may also differ from each other incoding sequence, resulting in additional differences in mRNA. Thesequence of the sense and antisense strands of the RNAi agent can thusbe tailored to correspond to that of an individual patient, if and whereneeded. RNAi agents can also be modified in sequence to reduceimmunogenicity, binding to undesired mRNAs (e.g., “off-target effects”)or to increase stability in the blood. These sequence variants areindependent of chemical modification of the bases or 5′ or 3′ or otherend-caps of the RNAi agents.

Lengths of Antisense and Sense Strands

The antisense and sense strands of RNAi agents can be of variouslengths, as described herein and as known in the art.

In one embodiment, each strand is a 49-mer or shorter.

Shorter lengths of siRNAs have been found to yield effective siRNAs. Forexample, each of the two RNA strands can be 19 to 25 nucleotides (nt),with a double-stranded region of 14-24 base pairs (bp), and at least one3′ end overhang of 1-5 nt. See, for example: U.S. Pat. Nos. 7,056,704and 7,078,196; Japanese JP 2002/546670; and European EP 1407044.Alternatively, the strand can each be 21 nt long, forming a 19 bp regionwith two 2 nt overhangs. Such a structure is defined herein as the“canonical” structure.

Alternatively, the strands can each be an 19-mer and together the twostrands can form a blunt-ended duplex.

Alternatively, the strands can each be an 18-mer and together the twostrands can form a blunt-ended duplex.

Alternatively, the sense strand can be significantly shorter than theantisense strand. In some embodiments, the antisense strand is about 21nt long, while the sense strand is only 15 or 16 nt long. Shortening thesense strand decreases the off-target effects mediated by the sensestrand being incorporated into RISC. Sun et al. 2008 Nature Biotech. 26:1379-1382; Chu and Rana. 2008 RNA 14: 1714-1719. The sense strand canbe, in various combinations, shortened, modified and/or 5′ end capped todecrease its RNAi activity.

Any length of either the antisense or sense strand can be combined withany other embodiment of a RNAi agent as described herein (e.g., any 3′end cap, modification, nucleotide replacement, 5′ end cap, overhang,delivery vehicle, etc.) as long as such combinations are not mutuallyexclusive (e.g., the presence or absence of an overhang may be dictatedby particular lengths of antisense and sense strands).

The 3′ end caps described herein thus can be used with any functionalRNAi agent with strand(s) of any length.

(Optional) Overhang(s)

The RNAi agents can also have overhangs of 0, 1, or 2 overhangs; in thecase of a 0 nt overhang, they are blunt-ended. A RNAi agent can thushave 0, 1 or 2 blunt ends. In a “blunt-ended RNAi agent” both strandsterminate in a base-pair; thus a blunt-ended molecule lacks either 3′ or5′ single-stranded nucleotide overhangs.

A 3′ end cap of the present disclosure can replace the functionality ofan overhang, or can be used in addition to an overhang on one or bothstrands.

As used herein, the term “overhang” or “nucleotide overhang” refer to atleast one unpaired nucleotide that protrudes from the end of at leastone of the two strands of the duplex structure of a RNAi agent. Theoverhang(s) may be on the 5′ and/or 3′ end of the sense and/or theantisense strand.

While both strands of the siRNA are generally RNA (although one or moreof the nucleotides can be replaced and/or modified), the overhang can beRNA or a variant thereof. Suitable overhangs include: RNA of anysequence or length (e.g., 1-5 nt), TT (a dithymidine dinucleotide) or UUor a variant thereof, such as dTdT, sdT, dTsdT, sdTsdT, or sdTdT, or thelike, which may be in either the inverted/reverse orientation or in thesame 5′ to 3′ orientation as the target gene specific sequence in theduplex.

Nucleotidic overhangs such as TT or UU do not recognize the target mRNAand are not considered part of the target sequence. Nonetheless, theoverhangs can be functional, as many canonical siRNAs do not function aswell without them. In addition, the overhangs provide some protection ofthe RNAi agent from degradation by nucleases, such as those in bloodserum or intestinal fluid. See: Elbashir et al. 2001 EMBO J. 23:6877-6888, especially FIG. 1F; Elbashir et al. 2001 Nature 411: 494-498;and Kraynack et al. 2006 RNA 12:163-176

As shown herein and as is known in the art, many attempts have been madeto replace overhangs with 3′ end caps to yield superior RNAi agents.These attempts, however, as detailed below, often yielded RNAi agentswhich were unable to both (1) mediate RNA interference and to (2) haveincreased resistance to nucleases and/or prolonged duration in bloodserum. In contrast, the 3′ end caps disclosed herein allow RNAinterference and increased duration of RNA interference activity in theserum.

Because they sometimes replace a dinucleotide overhang, a 3′ end cap(particularly a PAZ ligand) is in some documents referred to as a“dinucleotide surrogate”. However, it is noted that a 3′ end cap asdisclosed herein can also be used in addition to an overhang.

This document thus encompasses 3′ end caps, e.g., as shown in Tables 1and 2 and/or otherwise described herein, for use in RNAi agents.

(Optional) 5′ End Cap(s)

A “5′ cap” can be optionally attached at the 5′ end of the sense orantisense strand. The functions of the antisense and strands differ, asdo the structural requirements of the 5′ ends of these strands. A 5′ endcap on the antisense strand should not interfere with RNAi activitymediated by this strand; however, in some embodiments, the 5′ end cap onthe sense strand can interfere with RNAi activity mediated by the sensestrand. Either strand can be loaded into RISC, but only the antisensestrand targets the desired target. Loading of the sense strand can leadto off-target effects, e.g., RNA interference of an undesired target.Jackson et al. 2003 Nat. Biotech. 21: 635-637

In the case of the antisense strand: the 5′ end cap should not interferewith RNAi activity of this strand, but can provide at least someprotection (e.g., from nucleases such as those in serum or intestinalfluid). A 5′-phosphate on the guide strand is generally required foroptimal RNAi activity. A 5′ dT modification provides antisense strandstability and increases potency. Blocking of phosphorylation leads todecreased activity. In contrast, 1 to 3 ribonucleotides added to the 5′end improved inhibition. Morrissey et al. 2005 Nat. Biotech. 23:1002-1007. Some of the molecular interactions of the antisense strand 5′end with the Argonaute-2 (Ago2) component of RISC have been elucidated.Parker et al. 2005. Nature 434: 663-666; and Frank et al. 2010 Nature465: 818-822.

In contrast, in the case of the sense strand: a 5′ end cap that inhibitsRNA interference can be useful on this strand. As noted above, a5′-phosphate is generally required for optimal RNAi activity. Removal ofthe 5′-OH group is the simplest approach to prevent phosphorylation ofthe sense strand

In addition to 5′ end caps, other modifications or sets of modificationscan be used to reduce activity of the sense strand.

The 3′ end caps of the present disclosure can be used with any RNAiagent comprising a 5′ end cap on the sense strand and/or anymodification or set of modifications which reduces activity of the sensestrand.

(Optional) Additional Nucleotide Replacements and/or Modifications

The strands of a siRNA can generally comprise RNA molecules as expressedor found in nature (i.e., are naturally occurring), but alsonon-naturally occurring analogs and derivatives of nucleotidescomprising one or more ribonucleotide/ribonucleoside analogs orderivatives as described herein or as known in the art.

In some of the positions, the RNA nucleotides can be replaced by DNA, ora nucleotide of a different backbone, or PNA, LNA, Morpholino, TNA, GNA,ANA, HNA, CeNA, FANA, and/or UNA; and/or modified (including, but notlimited to, 2′-MOE, 2′-OMe, 2′-F, and 2′-H). In various embodiments, theRNAi agent can comprise one or more LNA which are at 5′ end and/or at 3′end (e.g., positions 18 and 19 in a 19-mer or positions 17 and 18 in an18-mer), and/or in the middle of a strand.

In some embodiments, the nucleotide replacements are in the last twobase-pairing nt (counting from 5′ to 3′), forming a clamp. A clampincludes without limitation a 2′-MOE clamp [wherein the last twobase-pairing nt (counting from 5′ to 3′) each have a 2′-MOEmodification]. Other variants of the clamp are possible, wherein, forexample, wherein the last two base-pairing nt (counting from 5′ to 3′)each are DNA, 2′-OMe, 2′-F or LNA, as shown in FIG. 20 C-E. It is notedthat the last two nt (counting from 5′ to 3′) can also be considered tobe the first two base-pairing nucleotides at the 3′ end of each strand(counting from 3′ to 5′). As shown herein and in U.S. Pat. No.8,084,600, the clamp can be on the antisense and/or sense strands.

Thus, while the nucleotides in each strand are generally RNA (meaningthat most of the nucleotides are RNA), some may be replaced by DNA ornucleotides of an alternative backbone such as peptide nucleic acids(PNA), locked nucleic acid (LNA), Morpholino, threose nucleic acid(TNA), and/or glycol nucleic acid (GNA). In some embodiments, only 1 or2 or 3 nt in one or both strands are replaced. In some embodiments, onlyabout 1-3 nt in one or both strands are replaced by DNA. Non-limitingexamples of this are shown in FIGS. 15B and 17A.

The RNA nucleotides in either strand can thus be replaced and/ormodified.

The RNA can be modified in the nucleobase structure or in theribose-phosphate backbone structure. However, in most embodiments, themolecules comprising ribonucleoside analogs or derivatives retains theability to form a duplex. As non-limiting examples, an RNA molecule canalso include at least one modified ribonucleoside, including but notlimited to a 2′-O-methyl modified nucleotide, a nucleoside comprising a5′ phosphorothioate group, a terminal nucleoside linked to a cholesterylderivative or dodecanoic acid bisdecylamide group, a locked nucleoside,an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholinonucleoside, an unlocked ribonucleotide (e.g., an acyclic nucleotidemonomer, as described in WO 2008/147824), a phosphoramidate or anon-natural base comprising nucleoside, or any combination thereof.Alternatively, an RNA molecule can comprise at least two modifiedribonucleosides, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 15, at least 20or more, up to the entire length of the dsRNA molecule. Themodifications need not be the same for each of such a plurality ofmodified ribonucleosides in an RNA molecule. In one embodiment, modifiedRNAs contemplated for use in methods and compositions described hereincomprise a 3′ end cap as disclosed herein and have the ability to formthe required duplex structure and that permit or mediate the specificdegradation of a target RNA via a RISC pathway.

Examples of modified nucleotides which can be used to generate the RNAiagent include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

A “modified variant” of a sequence disclosed herein includes any variantcomprising the same sequence, but with a modification in the base,sugar, phosphate or backbone (but not a base substitution, e.g., A forG, or C for U). Thus, a modified variant can comprise any modifiednucleotide described above (e.g., 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, etc.). When a base is replaced by acorresponding modified base (e.g., A for modified A), these modifiednucleotides do not constitute a mismatch or base difference. Thus agiven sequence with a U at a particular position and a modified variantcomprising a 5-fluorouracil, 5-bromouracil, 5-chlorouracil, or5-iodouracil at the same sequence would differ by 0 nt (or have nomismatches); however, a given sequence with a C at a particular positionand a different sequence with a 5-fluorouracil (wherein the twosequences are otherwise identical) would differ by 1 nt (1 mismatch).

In some embodiments, the RNAi agent according to the present inventionconfers a high in vivo stability by including a 3′ end cap and at leastone modified nucleotide in at least one of the strands. Thus the RNAiagent according to the present invention preferably contains at leastone modified or non-natural ribonucleotide. A lengthy description ofmany known chemical modifications are set out in published PCT patentapplication WO 200370918 and will not be repeated here. Suitablemodifications for oral delivery are more specifically set out in theExamples and description herein. Suitable modifications include, but arenot limited to modifications to the sugar moiety (i.e. the 2′ positionof the sugar moiety, such as for instance 2′-O-(2-methoxyethyl) or2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., analkoxyalkoxy group) or the base moiety (i.e. a non-natural or modifiedbase which maintains ability to pair with another specific base in analternate nucleotide chain).

Other modifications include so-called ‘backbone’ modificationsincluding, but not limited to, replacing the phosphoester group(connecting adjacent ribonucleotides with for instancephosphorothioates, chiral phosphorothioates or phosphorodithioates). Invarious embodiments, one or more phosphate group is replaced with:

In various additional embodiments, one or more phosphate group isreplaced by:

where R³ is selected from O⁻, S⁻, NH₂, BH₃, CH₃, C₁₋₆ alkyl, C₆₋₁₀ aryl,C₁₋₆ alkoxy and C₆₋₁₀ aryl-oxy, wherein C₁₋₆ alkyl and C₆₋₁₀ aryl areunsubstituted or optionally independently substituted with 1 to 3 groupsindependently selected from halo, hydroxyl and NH₂; and R⁴ is selectedfrom O, S, NH, or CH₂. Some of these replacement phosphate groups arealso shown in FIG. 18C.

In various embodiments, the phosphate of the phosphate group is replacedby arsenic (As), selenium (Se), or antimony (Sb). In one embodiment, thespacer is ribitol and no phosphate groups are replaced. In variousembodiments, the phosphate group is replaced by a sulfonamide group or acyano group or carboxamide. In various embodiments, the phosphate groupof the 3′ end cap is replaced by an arsenic, selenium, antimony orsulfonamide group or a cyano group or carboxamide. In variousembodiments, the phosphate group of the linker (e.g., C3, C4, or C6 orribitol, Diribitol, 2′-deoxyribitol, or 2′-methoxyethoxyribitol) isreplaced by an arsenic, selenium, antimony or sulfonamide group or acyano group or carboxamide.

W═O, S, NH, CH₂, . . . .

X₁, X₂═O⁻, S⁻, NH₂, BH₃ ⁻, CH₃, alkyl, aryl, O-alkyl, O-aryl, . . . .

Y═O, S, NH, CH₂, . . . . Z═C, Si, P, S, As, Se, Sb, Te, . . . .

R1=H, OH, F, NH₂, O-alkyl, O-aryl, O-alkyl-aryl, O-aryl-alkyl, NH-alkyl,N-dialkyl, . . . .R2=alkyl, aryl, alkyl-aryl, aryl-alkyl, . . . (PAZ ligand)BASE=H, adenine, cytosine, guanine, uracil, thymine, . . . .

Thus, the nucleotides of either or both strands of a RNAi agent usefulwith 3′ end caps disclosed herein can be replaced and/or modified.

(Optional) Patterns of Modifications

In some cases of modifying the nucleotides of a RNAi agent, themodifications are not random, but are arrayed in patterns. Thesepatterns (or schemes) increase the efficacy (RNAi activity), decreaseactivity of the sense strand or otherwise decrease off-target effects,reduce degradation or immunogenicity, and/or increase the biologicalhalf-life (e.g., time of duration of activity) of the RNAi agent.

In one pattern of modification, multiple positions of the sense strandare 2′-MOE. As a non-limiting example, most or all of the pyrimidinesare 2′MOE in the sense strand. Modifying more than half of the positionsin a sense strand with 2′-MOE can decrease activity. When all thepositions of the sense strand are 2′-MOE often abolishes activity.

Various patterns of modifications are shown in FIGS. 7, 11, 14, 15A,15B, and 17A.

FIG. 15A (top) shows a non-limiting example of the arrangement of apattern of 2′-OMe and 2′-MOE modifications in a 19-mer blunt-ended RNAiagent. In the example, the 3′ end cap shown is C3, but other 3′ end capscan be used with this modification pattern (e.g., those disclosedherein). This modification pattern also includes a MOE clamp (whereinthe last two base-pairing nucleotides counting from 5′ to 3′ have 2′-MOEmodifications). The last two nt counting from 5′ to 3′ can also beconsidered to be the first two base-pairing nucleotides at the 3′ end ofeach strand (counting from 3′ to 5′).

FIG. 15A (bottom) shows a non-limiting example of a modification patternusing 2′F modifications. Again, in this example, the 3′ end cap shown isC3, but other 3′ end caps can be used with this modification scheme(e.g., those disclosed herein).

FIG. 15B (top) shows a “wt” (“wild-type”) siRNA and a correspondingnon-limiting example modification scheme of this siRNA. The examplemodified siRNA has 2′-OMe and phosphorothioate (s).

FIG. 15B (bottom) shows non-limiting examples of modification schemesfor the canonical 21-mer siRNA, and for the 18- or 19-mer formats. Inthese schemes, “L” indicates the 3′ end cap (e.g., a PAZ Ligand).

In various other modification patterns, the RNAi agent comprises atleast one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein theuridine is a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′(5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide; at least one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide,wherein the 5′-cytidine is a 2′-modified nucleotide; and/or at least one5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide.

Other patterns of modifications can be used with any RNAi agentcomprising a 3′ end cap as disclosed herein.

Particularly preferred modification patterns include but are not limitedto:

All 3′ overhangs as 2′-OMe-U 2′-OMe-U

A85: All U as 2′-OMe-U, except pos. 1, 2 and 14

S26: All U as 2′-OMe-U and all C as 2′-OMe-C

A51: All U as 2′-OMe-U and all C as 2′-OMe-C, except pos. 1, 2 and 14

S26: All U as 2′-OMe-U and all C as 2′-OMe-C

A48: UA as 2′-OMe-U A and all CA as 2′-OMe-C A, first 5′-N is DNA

S26: All U as 2′-OMe-U and all C as 2′-OMe-C

The 3′ end caps disclosed herein can thus be used with any RNAi agent,wherein at least one nucleotide of at least one strand of the RNAi agenthas been replaced and/or modified, and wherein the modification(s) ofthe nucleotide(s) can be arrayed in a pattern(s) of modification.

In various patterns of modification, the pattern comprises a 2′-MOEclamp [wherein the last two base-pairing nt (counting from 5′ to 3′)each have a 2′-MOE modification]. Other variants of the clamp arepossible, wherein, for example, wherein the last two base-pairing nt(counting from 5′ to 3′) each are DNA, 2′-OMe, 2′-F or LNA, as shown inFIG. 20 C-E. It is noted that the last two nt (counting from 5′ to 3′)can also be considered to be the first two base-pairing nucleotides atthe 3′ end of each strand (counting from 3′ to 5′). As shown herein andin U.S. Pat. No. 8,084,600, the clamp can be on the antisense and/orsense strands.

Any embodiments of any RNAi agent described herein can be combined withany other embodiment, provided that the embodiments are not mutuallyexclusive (e.g., a single RNAi agent cannot simultaneously have bothexactly 0 and exactly 2 overhangs).

Thus, the 3′ end caps disclosed herein can be used with any RNAi agentas described herein or as known in the art, wherein the strands of theRNAi agent are of any length, the RNAi agent can comprise 0, 1, or 2overhangs or 0, 1 or 2 blunt ends, one or more nucleotides of one orboth stands can be replaced or modified, and the modification(s) can bearrayed in a pattern(s) or scheme(s) of modification, and the antisenseand/or sense strand can comprise a 5′ end cap, wherein the 5′ end cap ofthe sense strand (if present) reduces RNA interference activity mediatedby the sense strand.

The 3′ end caps disclosed herein can also be used with any additionalRNAi agent format or structure disclosed herein or known in the art.

Additional RNAi Agents

In additional to the structures listed above, additional types ofmolecules have been devised which are also capable of mediating RNAinterference. In these structures, the strands are not necessarily RNA,and the strands can be can be longer or shorter than the canonical,and/or blunt-ended, and/or comprise one or more modification, mismatch,gap, and/or nucleotide replacement.

The term “RNAi agent” is intended to encompass any molecule describedherein or known in the art capable of mediating RNA interference,including, without limitation, siRNA (whether of canonical or otherstructure), or any other molecule capable of mediating RNA interference.The 3′ end caps described herein can be used with any RNAi agent.

Thus, the 3′ end caps disclosed herein can be used on any RNAi agent(including siRNA) or on any other RNAi agent, including, inter alia, andwithout limitation:

shRNA (small hairpin RNA or short hairpin RNA), which comprises asequence of RNA that makes a tight hairpin turn and, like siRNAs,silences targets via RISC. The antisense and sense strand are thusconnected by a hairpin. shRNAs can be expressed, for example, viadelivery of plasmids or through viral or bacterial vectors. Variousvarieties of shRNAs are known in the art. See, for example: Xiang et al.2006. Nature Biotech. 24: 697-702; Macrae et al. 2006 Science 311:195-8. Lombardo et al. 2007. Nature Biotech. 25: 1298-1306; Wang et al.2011. Pharm. Res. 28: 2983-2995; Senzer et al. 2011. Mol. Ther. 20:679-686.

miRNA (microRNA), which is a small RNA molecule (ca. 22 nt) that, likesiRNAs, also silences targets via RISC. Naturally-occurring miRNAs areencoded by eukaryotic nuclear DNA; miRNAs are generated bypost-transcriptional RNA processing, and function via base-pairing withcomplementary sequences within mRNA molecules, usually resulting intranslational repression or target degradation and gene silencing. Thehuman genome can encode over 1000 miRNAs, which may target about 60% ofmammalian genes and are abundant in many human cell types. Variousvarieties of naturally-occurring and artificial derivatives of miRNAsare known in the art. See, for example: Lewis et al. 2003. Cell 115:787-798; Lim et al. 2003. Genes Dev. 17: 991-1008; He et al. 2004. Nat.Rev. Genet. 5: 522-31; Bentwich et al. 2005. Nat. Genet. 37: 766-70;Lewis et al. 2005. Cell 120: 15-20; Kusenda et al. 2006. Biomed Pap MedFac Univ Palacky Olomouc Czech Repub 150: 205-15; Zhang et al. 2006. J.Gen. Gen. 36: 1-6; Brodersen et al. 2008. Science 320: 1185-90; Friedmanet al. 2009. Genome Res. 19 (1): 92-105; Bartel 2009. Cell 136 (2):215-33.

sisiRNA (small internally segmented interfering RNA), wherein the sensestrand comprises at least one single-stranded nick. This nick decreasesthe incorporation of the sense strand into the RISC complex and thusreduces off-target effects. See: WO 2007/107162.

DNA-RNA chimera, wherein the seed portion of each strand is DNA, whilethe remainder of each strand is RNA. See: Yamato et al. 2011. CancerGene Ther. 18: 587-597.

siRNA comprising two mismatches, wherein that the molecule comprisesthree short double-stranded regions. In one embodiment of this RNAiagent, the guide (antisense) strand is a 22-mer, while the sense strandis a 20-mer (producing only a single 2-nt overhang on the 3′ end of theanti-sense strand; and two mismatches produce double-stranded regions of6, 8 and 4 bp. See: U.S. Pat. App. 2009/0209626

aiRNA (assymetrical interfering RNA), wherein the sense strand isshorter than 19-nt long, so that the anti-sense strand is preferentiallyloaded into RISC, and thus off-target effects are reduced. In variousembodiments of this RNAi agent, the anti-sense strand is 21-nt long, butthe sense strand is only 15 or 16 nt long. See: Sun et al. 2008 NatureBiotech. 26: 1379-1382; and Chu and Rana. 2008 RNA 14: 1714-1719.

Thus, any 3′ end cap disclosed herein can be used with any of thevarious formats of RNAi agents described above or otherwise known in theart, including siRNAs (including but not limited to those of thecanonical structure), shRNAs, miRNAs, sisiRNAs, DNA-RNA chimeras, siRNAscomprising two mismatches (or more mismatches), or aiRNAs.

3′ End Caps

The RNAi agent of the present disclosure comprises a 3′ end cap. Theterms “3′ end cap”, “3′ end cap modification”, “end cap”, “Cap”, “3′ endmodification” and the like include a chemical moiety attached to the endof a double-stranded nucleotide duplex, but is used herein to exclude achemical moiety that is a nucleotide or nucleoside. A “3′ end cap” isattached at the 3′ end of a nucleotide or oligonucleotide (e.g., is amodification at the 3′ carbon of the 3′ nucleotide at the 3′ terminus ofat least one strand) and protects the molecule from degradation, e.g.,from nucleases, such as those in blood serum or intestinal fluid. “3′end caps” include but are not limited to “PAZ ligands,” which termincludes 3′ end caps which interact with the PAZ domain of the enzymeDicer. 3′ end caps are sometimes referred to as “non-nucleotide overhangmimics” or “LMW mimics of dinucleotide overhangs” or the like.

This disclosure notes that some documents refer to a 3′ end cap asdescribed herein (e.g., X109 or X110 or X111, etc.) as an “overhang” ora “3′ overhang”; however, this document differentiates a 3′ end cap froman “overhang” and uses the term “overhang” only to refer to anucleotidic overhang (e.g., one comprising only nucleotides such as A,C, G, U or T, such as UU or TT). Thus, as defined herein, a “3′ end cap”is not an overhang.

As defined herein, a 3′ end cap can be used in place of or in additionto an overhang (i.e., a nucleotidic overhang). Earlier work withcanonical siRNA structures suggested that the 2-nt overhang was usefulfor RNA interference activity, while blunt-ended dsRNAs (lacking theoverhangs) were generally not effective. See, for example, Elbashir etal. 2001 EMBO J. 23: 6877-6888, especially FIG. 1F. However, dsRNA, evenwith the overhangs, were subject to enzymatic degradation. As notedelsewhere by the Applicants, “unmodified siRNAs are subject to enzymaticdigestion, mainly by nucleases.” (WO 2007/128477, page 1). 3′ end capswere thus designed to perform several functions, including (1) allowingthe molecule to mediate RNA interference activity, and (2) protectingthe molecule from degradation.

It is noted, though, that the 3′ end caps disclosed herein can be usedin addition to as well as in place of 3′ overhangs.

Because a 3′ end cap can be used instead of an overhang such as UU orTT, the 3′ end caps described herein are sometimes referred to as“3′-Dinucleotide surrogates”.

A few 3′ end caps have been disclosed for use with siRNAs. It is notedthat of the 3′ end caps which have been described chemically, many ofthese have been shown not to be functional. A functional 3′ end cap canbe able to perform these functions: (1) allow the double-stranded RNA tofunction in RNA interference; and (2) increase the stability of themolecule, e.g., by protecting it from nucleases, such as those found inblood serum or intestinal fluid.

Non-Functional 3′ End Caps

Many 3′ end caps described in the literature are unable to perform bothof these functions. In some cases, the placement of the end caps isimportant; some end caps may be functional when placed on only onestrand, but not functional if placed on both strands and/or on both 5′and 3′ ends of both strands.

It is impossible to predict which 3′ end caps will perform bothfunctions without experimentation. In fact, while many endcaps werepredicted to be suitable for RNA interference (e.g., in US2003/0143732), many later were discovered not to perform both functions.

Other scientists have empirically found that, despite predictions, someendcaps or overhangs (1) stabilized the siRNA but (2) did NOT allow RNAiactivity. For example, the TT (dithymidine) in combination with 2′-OMemodifications at all positions, Czauderna et al. 2003 Nucl. Acids Res.31:2705-2716, FIG. 4B. Hadwiger et al. also note that complete2′-O-methylation rendered the siRNA serum nuclease-resistant, althoughgene silencing activity was almost completely abolished. Hadwiger et al.2005, pages 194-206, in RNA Interference Technology, ed. K. Appasani,Cambridge University Press, Cambridge, UK.

Other endcaps or overhangs (1) did NOT stabilize the siRNA, though (2)they did allow RNAi activity. For example, the TT at both 3′ ends orboth 5′ ends of a siRNA. Czauderna et al. 2003, FIG. 4B.

Still other endcaps (1) did NOT stabilize the siRNA AND (2) did NOTallow RNAi activity such examples include: the amino-C6 linker orinverted abasic nucleotide. Czauderna et al. 2003, FIG. 4B.

Additional examples of 3′ end caps which are non-functional under atleast some conditions include:

Inverted (deoxy) a basics, which were neither stabilize siRNA nor allowsiRNA activity when present on both 5′ and both 3′ ends. See: Czaudernaet al. 2003 Nucl. Acids Res. 31:2705-2716, FIG. 4B.

Modified base nucleotides such as 5-propynyl-U, which do not bothstabilize the siRNA and allow RNAi activity. Deleavey et al. 2009 Curr.Prot. Nucl. Acid Chem. 16.3.1-16.3.22; Terrazas et al. 2009 NucleicAcids Res. 37: 346-353.

At least some amino-substituted lower alkyls, including aminohexylphosphate, which was not able to stabilize the siRNA. When present onboth 5′ ends and both 3′ ends, it prevented RNAi activity. See:Czauderna et al. 2003, FIG. 4B.

Fluoroscein (e.g., a fluorescent chromophore), which was found toinhibit RNA interference activity when conjugated to the 3′ end of theantisense strand. The sense strand can tolerate, for example, aconjugation of fluorescein at the 3′-end, but the antisense strandcannot. Harboth et al. 2003 Antisense Nucl. Acid Drug Dev. 13: 83-105.See: Harboth et al. 2003 Antisense Nucl. Acid Drug Dev 13: 83-105.

Cyanine (e.g., Cy5), which is non-functional. See: Song et al. 2003Nature Med. 9: 347-351. See page 347, second col.

3′ phosphate as a 3′ end cap, suggested by U.S. Pat. No. 5,998,203(paragraph [017]), but later shown not to both stabilize the 3′ end of asiRNA and allow RNAi activity, Schwarz et al. 2002 Mol. Cell 10:537-548; and Lipardi et al. 2001 Cell 107: 299-307.

3′-aminopropylphosphoester, which reduced RNA interference activity.See: Schwarz et al. 2002 Mol. Cell 10: 537-548, FIG. 2.

Thus, not all moieties tested as 3′ end caps are capable of bothallowing RNA interference and protecting the molecule from degradation.

Functional 3′ End Caps

In contrast to the non-functional 3′ end caps and overhangs describedabove, functional 3′ end caps are described in, for example, U.S. Pat.Nos. 8,097,716; 8,084,600; 8,344,128; 8,404,831; and 8,404,832. Thesedisclose functional 3′ end caps comprising a phosphate and nicknamed asC3, C6, C12, Triethylene glycol, Cyclohexyl (or Cyclohex), Phenyl,Biphenyl, Adamantane and Lithocholic acid (or Lithochol).

These functional 3′ end caps are diagrammed below, wherein they areshown bonded to a phosphate:

It is noted that the terminology used in the present disclosure differsslightly from that used in U.S. Pat. Nos. 8,097,716; 8,084,600;8,344,128; 8,404,831; and 8,404,832. In various embodiments, the presentdisclosure pertains to RNAi agents comprising a first strand and asecond strand, wherein, in some embodiments, the 3′ end of the firstand/or second strand terminates in a phosphate (or modifiedinternucleoside linker) and further comprises a 3′ end cap. In thediagrams directly above, the phosphate and the 3′ end cap are shown.

The 3′ end caps disclosed in U.S. Pat. Nos. 8,097,716; 8,084,600;8,344,128; 8,404,831; and 8,404,832 were superior to those which weredevised before them. For example, unlike other possible endcaps, thesewere able to both protect the siRNAs from degradation (e.g., fromnucleases, such as in blood or intestinal fluid), and also allow RNAinterference.

However, many of the novel 3′ end caps of the present disclosure (e.g.,those listed in Tables 1 and 2) are even further improved. For example,siRNAs with X058 (as disclosed herein) show a higher duration ofactivity than a siRNA with C6 (FIG. 22). HuR siRNAs with X058 showedgreater efficacy at Day 7 and at Day 10 in Huh-7 cells.

Various novel 3′ end caps disclosed herein include those designated asPAZ ligands, as they interact with the PAZ domain of Dicer.

PAZ Ligands

As noted above, when a long dsRNA molecule is introduced into a cell,Dicer chops the dsRNA is shorter segments called siRNAs. A homologue ofDicer is common to all organisms in which dsRNA-mediated gene silencinghas been observed. Myers et al. 2005. In RNA Interference Technology,ed. Appasani, Cambridge University Press, Cambridge UK, p. 29-54;Bernstein et al. 2001 Nature 409: 363-366; and Schauer et al. 2002Trends Plant Sci. 7: 487-491. Dicer is an RNase III enzyme and iscomposed of six recognizable domains. At or near the N-terminus is anapprox. 550 aa DExH-box RNA helicase domain, which is immediatelyfollowed by a conserved approx. 100 aa domain called DUF283. JustC-terminal to DUF283 domain is the PAZ (for Piwi/Argonaute/Zwille)domain. The domain recognizes single stranded dinucleotide overhangs.Lingel et al. 2003 Nature 426: 465-469; Song et al. 2003 Nature Struct.Biol. 10: 1026-1032; Yan et al. 2003 Nature 426: 468-474; Lingel et al.2004 Nature Struct. Mol. Biol. 11: 576-577; Ma et al. 2004 Nature 429:318-322. Presumably, the PAZ domain in Dicer could also bind RNA toposition the catalytic domains for cleavage. Zhang et al. 2004 Cell 118:57-68. The C-terminus of the Dicer protein is composed of two RNAse IIIcatalytic domains and a putative dsRNA-binding domain.

Table 2 lists various 3′ end caps, including many PAZ ligands.

Arrangement and Non-Identical Nature of 3′ End Caps

The anti-sense and sense strands are biochemically distinct. As notedabove, the antisense strand is preferably loaded into RISC, as thisstrand targets the desired target. Incorporation of the sense strand canlead to off-target effects.

It is known that some 3′ end caps can be more useful on one strand thanon the other. For example, as noted above, The sense strand cantolerate, for example, a conjugation of fluorescein at the 3′-end, butthe antisense strand cannot. Harboth et al. 2003 Antisense Nucl. AcidDrug Dev. 13: 83-105.

An RNAi Agent Comprising a 3′ End Cap Described Herein

In one particular specific embodiment, the present disclosure relates toa composition comprising a RNAi agent comprising a first strand and asecond strand, wherein the first or second strand comprise a 3′ end capselected from the 3′ end caps listed in Table 2. In one embodiment, thecomposition comprises a RNAi agent comprising a first and an secondstrand, wherein the first and second strand comprise a 3′ end capselected from the 3′ end caps listed in Table 2. Thus, in short: In oneembodiment, the composition comprises a RNAi agent comprising a firstand an second strand, wherein the first and/or second strand comprise a3′ end cap selected from the 3′ end caps listed in Table 2. In someembodiments, the first and second strand are the anti-sense and sensestrands, respectively. In some embodiments, the first and second strandsare the sense and anti-sense strands, respectively.

A RNAi agent is a double-stranded molecule capable of mediating RNAinterference, including but not limited to siRNAs.

Various specific embodiments of this embodiment are described below.

In one embodiment, the composition further comprises a second RNAiagent. In various embodiments, the second RNAi agent is physicallyseparate from the first; or the two RNAi agents are physically connected(e.g., covalently linked or otherwise conjugated) or combined in thesame pharmaceutical composition, or are both elements in the sametreatment regimen.

In one embodiment, the antisense strand is about 30 or fewer nt inlength.

In one embodiment, the sense strand and the antisense strand form aduplex region of about 15 to about 30 nucleotide pairs in length.

In one embodiment, the antisense strand is about 15 to about 36 nt inlength, including about 18 to about 30 nt in length, and furtherincluding about 19 to about 21 nt in length and about 19 to about 23 ntin length. In one embodiment, the antisense strand has at least thelength selected from about 15 nt, about 16 nt, about 17 nt, about 18 nt,about 19 nt, about 20 nt, about 21 nt, about 22 nt, about 23 nt, about24 nt, about 25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 ntand about 30 nt.

In one embodiment, the 3′ end cap causes the RNAi agent to haveincreased stability in a biological sample or environment, e.g.,cytoplasm, interstitial fluids, blood serum, lung or intestinal lavagefluid.

In one embodiment, the RNAi agent further comprises at least one sugarbackbone modification (e.g., phosphorothioate linker) and/or at leastone 2′-modified nucleotide. In one embodiment, all the pyrimidines are2′ O-methyl-modified nucleotides.

In one embodiment, the RNAi agent comprises: at least one5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide, wherein the uridine is a2′-modified nucleotide; and/or at least one 5′-uridine-guanine-3′(5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide; and/or at least one 5′-cytidine-adenine-3′ (5′-ca-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide;and/or at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide,wherein the 5′-uridine is a 2′-modified nucleotide.

In one embodiment, the RNAi agent comprises a 2′-modification selectedfrom the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and2′-O—N-methylacetamido (2′-O-NMA). In one embodiment, all thepyrimidines are 2′ O-methyl-modified nucleotides.

In one embodiment, the RNAi agent comprises a blunt end.

In one embodiment, the RNAi agent comprises an overhang having 1 to 4unpaired nucleotides.

In one embodiment, the RNAi agent comprises an overhang at the 3′-end ofthe antisense strand of the RNAi agent.

In one embodiment, the RNAi agent is ligated to one or more diagnosticcompound, reporter group, cross-linking agent, nuclease-resistanceconferring moiety, natural or unusual nucleobase, lipophilic molecule,cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin,terpene, triterpene, sarsasapogenin, Friedelin,epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate,dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligolactate 15-mer, natural polymer, low- or medium-molecular weightpolymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-bindingagent, integrin-targeting molecule, polycationic, peptide, polyamine,peptide mimic, and/or transferrin.

In one embodiment, the RNAi agent is capable of inhibiting expression oftarget gene by at least about 60% at a concentration of 10 nM in cellsin vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression oftarget gene by at least about 70% at a concentration of 10 nM in cellsin vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression oftarget gene by at least about 80% at a concentration of 10 nM in cellsin vitro.

In one embodiment, the RNAi agent is capable of inhibiting expression oftarget gene by at least about 90% at a concentration of 10 nM in cellsin vitro.

In one embodiment, the RNAi agent has an EC50 of no more than about 0.1nM in cells in vitro.

In one embodiment, the RNAi agent has an EC50 of no more than about 0.01nM in cells in vitro.

In one embodiment, the RNAi agent has an EC50 of no more than about0.001 nM in cells in vitro.

Pharmaceutical Compositions of a RNAi Agent to Target Gene

In one particular specific embodiment, the present disclosure relates toa composition comprising a RNAi agent comprising a first and an secondstrand, wherein the first and/or second strand comprise a 3′ end capselected from the 3′ end caps listed in Table 2, wherein the compositionis in a pharmaceutically effective formulation.

In one embodiment, the present disclosure pertains to the use of a RNAiagent in the manufacture of a medicament for treatment of a targetgene-related disease, wherein the RNAi agent comprises a sense strandand an antisense strand, wherein the antisense strand comprises at least15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides fromthe antisense strand of a RNAi agent to target gene selected from thosespecific duplex provided herein and as listed, e.g., in Table 2.

In one embodiment, the pharmaceutical composition comprises a deliveryvehicle and a RNAi agent comprising a 3′ end cap.

Other modifications known to one skilled in the art are contemplated asbeing encompassed within the invention. Exemplary modifications include,but are not limited to, the presence of gaps or mismatches between thebase pairs in the sense and antisense strands, the presence of nicks orbreaks in the internucleoside linkages in the sense strand, and thelike.

Pharmaceutical Compositions

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. Formulations for oral use can also bepresented as hard gelatin capsules wherein the active ingredient ismixed with an inert solid diluent, for example, calcium carbonate,calcium phosphate or kaolin, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin or olive oil. Aqueous suspensions containthe active materials in a mixture with excipients suitable for themanufacture of aqueous suspensions.

Oral administration of the compositions of the invention include allstandard techniques for administering substances directly to the stomachor gut, most importantly by patient controlled swallowing of the dosageform, but also by other mechanical and assisted means of such delivery.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient. It is understood that the specific dose level for anyparticular subject depends upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

Therapeutic effect of the therapeutic agents of the invention may beenhanced by combination with other agents. Typically such other agentswill include agents known for use in treating similar diseases, such asangiogenic disorders.

The RNAi agents of the invention and formulations thereof can beadministered orally, topically, parenterally, by inhalation or spray, orrectally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and/or vehicles. Theterm parenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, intraperitoneal, orintrathecal injection, or infusion techniques and the like. Where two ormore different RNAi agents are administered, each may be administeredseparately or co-administered. Where each is administered separately,the method and/or site of administration may be the same or different,e.g., both RNAi agents may be administered intravenously orsubcutaneously, or a first RNAi agent may be administered intravenouslywith a second Rai agent administered subcutaneously, etc.

In various embodiments, the disclosure encompasses a composition orpharmaceutical composition comprising a RNAi agent, wherein one or bothstrands comprises a 3′ end cap, the composition further comprising ahelper lipid, a neutral lipid, and/or a stealth lipid.

In various embodiments, the composition further comprises a helperlipid.

In various embodiments, the composition further comprises a neutrallipid.

In various embodiments, the composition further comprises a stealthlipid.

In various embodiments, the helper lipid, neutral lipid and stealthlipid are selected from those disclosed in: published patent app. US2011-0200582. Additional compositions that can be used for delivery ofthe various RNAi agents are known in the art, e.g., are provided in U.S.Applications No. 61/774,759; 61/918,175, filed Dec. 19, 2013;61/918,927; 61/918,182; 61/918941; 62/025224; 62/046487; andInternational Applications No. PCT/US04/042911; PCT/EP2010/070412;PCT/IB2014/059503.

In various embodiments, the composition further comprises an additionalbiologically active agent.

In various embodiments, the helper lipid is cholesterol and thebiologically active agent is a siRNA.

In various embodiments, the composition is in the form of a lipidnanoparticle.

A Method of Treatment Using a RNAi Agent Described Herein

In one particular specific embodiment, the present disclosure relates toa method of treating a target gene-related disease in an individual,comprising the step of administering to the individual a therapeuticallyeffective amount of a composition comprising a RNAi agent comprising afirst strand and a second strand, wherein the first and/or second strandcomprise a 3′ end cap selected from the 3′ end caps listed in Table 2.In one particular specific embodiment, the present disclosure relates toa method of inhibiting the expression of target gene in an individual,comprising the step of administering to the individual a therapeuticallyeffective amount of a composition comprising a RNAi agent of the presentdisclosure.

In one embodiment of the method, the composition further comprises apharmaceutically effective formulation.

Various particular specific embodiments of these embodiments aredescribed below.

In one embodiment, the method further comprises the administration of anadditional treatment. In one embodiment, the additional treatment is atherapeutically effective amount of a composition.

In one embodiment, the additional treatment is a method (or procedure).

In one embodiment, the additional treatment and the RNAi agent can beadministered in any order, or can be administered simultaneously.

In one embodiment, the method further comprises the step ofadministering an additional treatment for the disease.

In one embodiment, the method further comprises the step ofadministering an additional treatment or therapy selected from the listof an additional antagonist to a target gene-related disease.

In one embodiment, the composition comprises a second RNAi agent totarget gene. In various embodiments, the second RNAi agent is physicallyseparate from the first, or the two are physically connected (e.g.,covalently linked or otherwise conjugated).

OTHER EMBODIMENTS

Various particular specific embodiments of this disclosure are describedbelow.

In one embodiment, the disclosure pertains to a composition according toany of the embodiments described herein, for use in a method of treatinga target gene-related disease in an individual, the method comprisingthe step of administering to the individual a therapeutically effectiveamount of a composition according to any of the claims.

One embodiment of the disclosure is the use of a composition accordingto any of these embodiments, in the manufacture of a medicament fortreatment of an target gene-related disease.

In one embodiment, the disclosure pertains to the composition of any ofthe above embodiments, for use in the treatment of an targetgene-related disease.

ADDITIONAL DEFINITIONS

Unless defined otherwise, the technical and scientific terms used hereinhave the same meaning as that usually understood by a specialistfamiliar with the field to which the present disclosure belongs.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein.

Claims to the present disclosure are non-limiting and are providedbelow.

Although particular embodiments and claims have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims, or the scope of subject matter ofclaims of any corresponding future application. In particular, it iscontemplated by the inventors that various substitutions, alterations,and modifications may be made to the present disclosure withoutdeparting from the spirit and scope of the present disclosure as definedby the claims.

The choice of nucleic acid starting material, clone of interest, orlibrary type is believed to be a matter of routine for a person ofordinary skill in the art with knowledge of the embodiments describedherein. Other aspects, advantages, and modifications considered to bewithin the scope of the following claims. Redrafting of claim scope inlater-filed corresponding applications may be due to limitations by thepatent laws of various countries and should not be interpreted as givingup subject matter of the claims.

Various additional formulations and obvious variants of the described 3′end caps can be devised by those of ordinary skill in the art.Non-limiting example RNAi agents wherein one or both strands comprises a3′ end cap are described in the Examples below, which do not limit thescope of the present disclosure as described in the claims.

Examples Example 1 Serum Stability of siRNAs with 3′ End Caps

The efficacy of a variety of different 3′ end caps (3′-terminaloverhangs) was tested. 10 siRNAs were prepared with an identicalsequence (mF7-III target gene, 19-mer blunt-ended, A12S17 modificationscheme)

10 different non-nucleotidic 3′-terminal caps were used.

These were tested in mouse and human sera at 4 time points ParentmF7-III in A6S11 format and wt (wild-type) luc (luciferase) siRNAs wereused as controls

The molecules used are diagrammed in FIG. 1.

Table 5 below provides the sequences for these molecules.

TABLE 5 For- For- siRNA mat siRNA passenger mat ID Project Serum sense sequence anti siRNA guide sequence 144033 mFVII  Mouse S17uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps 5 C3 5 C3149853 mFVII  Mouse S17 uGu cuu GGu uuc AAu uA5  A12UUu AAU UGA AAC cAA GA6  3′-caps 5 C6 5 C6 149855 mFVII  Mouse S17uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps 5 C125 C12 149857 mFVII  Mouse S17 uGu cuu GGu uuc AAu uA5  A12UUu AAU UGA AAC cAA GA6  3′-caps 5 glycol 5 glycol 149859 mFVII  MouseS17 uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps5 cyclohex 5 cyclohex 149861 mFVII  Mouse S17 uGu cuu GGu uuc AAu uA5 A12 UUu AAU UGA AAC cAA GA6  3′-caps 5 phenyl 5 phenyl 149863 mFVII Mouse S17 uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps5 biphenyl 5 biphenyl 149865 mFVII  Mouse S17 uGu cuu GGu uuc AAu uA5 A12 UUu AAU UGA AAC cAA GA6  3′-caps 5 lithochol 5 lithochol 149867mFVII  Mouse S17 uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6 3′-caps 5 amino C7 5 amino C7 149869 mFVII  Mouse S17uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps 5 amino C35 amino C3   8548 Luc   Mouse S0 TCGAAGTACTCAGCGTAAGTT A0CTTACGCTGAGTACTTCGATT stability ctrl. 144049 mFVII  Mouse S1uGu cuu GGu uuc AAu uAA  A1 UUu AAU UGA AAC cAA GAc  w/o cap AdTsdTAdTsdT 144033 mFVII  Human S17 uGu cuu GGu uuc AAu uA5  A12UUu AAU UGA AAC cAA GA6  3′-caps 5 C3 5 C3 149853 mFVII  Human S17uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps 5 C6 5 C6149855 mFVII  Human S17 uGu cuu GGu uuc AAu uA5  A12UUu AAU UGA AAC cAA GA6  3′-caps 5 C12 5 C12 149857 mFVII  Human S17uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps 5 glycol5 glycol 149859 mFVII  Human S17 uGu cuu GGu uuc AAu uA5  A12UUu AAU UGA AAC cAA GA6  3′-caps 5 cyclohex 5 cyclohex 149861 mFVII Human S17 uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps5 phenyl 5 phenyl 149863 mFVII  Human S17 uGu cuu GGu uuc AAu uA5  A12UUu AAU UGA AAC cAA GA6  3′-caps 5 biphenyl 5 biphenyl 149865 mFVII Human S17 uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps5 lithochol 5 lithochol 149867 mFVII  Human S17 uGu cuu GGu uuc AAu uA5 A12 UUu AAU UGA AAC cAA GA6  3′-caps 5 amino C7 5 amino C7 149869 mFVII Human S17 uGu cuu GGu uuc AAu uA5  A12 UUu AAU UGA AAC cAA GA6  3′-caps5 amino C3 5 amino C3 144049 mFVII w/o   Human NA NA A12UUu AAU UGA AAC cAA GAc  cap anti AdTsdT 144053 mFVII w/o   Human S17uGu cuu GGu uuc AAu uAA  NA NA cap sense AdTsdT

The sense sequences are represented from top to bottom by SEQ ID NOs: 92to 114; the guide (anti-sense) sequences are represented from top tobottom by SEQ ID NOs: 115 to 137, The 3′ end caps used in this exampleare diagrammed below, in the context of the RNAi agent strang:

Materials and Methods:

RNA samples were incubated in 100% mouse serum and human serum at 37°C., withdrawn at 0, 5′, 6 h and 24 h time points and snap-frozen. Oligoswere separated by precast hydrogels (Elchrom Scientific) and visualizedwith SYBR gold (Biorad, Chemidoc XRS).

FIG. 2 shows the efficacy of various 3′ end caps described in Example 1in allowing the RNAi agent to mediate RNA interference. All of the 3′end caps—C3, C6, C12, Triethylene glycol, Cyclohexyl, Phenyl, Biphenyl,Adamantane and Lithocholic acid—allow the RNAi agent to perform RNAinterference.

FIG. 3 shows the efficacy of various 3′ end caps described in Example 1in reducing and/or preventing nuclease degradation in serum.

In mouse serum all 3′-capped A12S17 siRNAs display high resistance up to24 h.

In human serum C3, C12 and lithochol appear to be less stable ascompared to the other derivatives. However, in both experiments, C3,biphenyl and litochol display significantly weaker bands as compared tothe other derivatives. However, there is a need to clarify whether thisis due to lower synthesis/dsRNA quality (as indicated by gel-based QC)or due to a technical gel-based artifact (lithocholic acid may stick tohuman serum and thus gets protected from SYBR GOLD intercalation).

Single-strand antisense A12 is degraded rapidly in human serum whereasthe parent sense S17 strand (with more chemical modifications) resists abit longer but not as long as the dsRNA. Enzymatic stability correlateswith thermal dsRNA stability.

Thus, this Example shows that siRNAs with these various 3′ end caps wereable to mediate RNA interference against FVII (Factor VII). The 3′ endcap modifications designated as C3, C6, C12, glycol, cyclohex, phenyl,biphenyl, lithochol, C7 amino and C3 amino showed increased stability inmouse serum at 1′,30′, 6 h and 24 hrs compared to luciferase and dTsdTcontrols. Those 3′-end modifications designated C3, C6, glycol,cyclohex, phenyl and biphenyl, C7 amino and C3 amino also showedincreased stability in human serum compared to controls.

Example 2 The Synthesis of Various 3′ End Cap Succinate Esters andAlcohols are Presented Below

Example 2.A X027 succinate ester Example 2.B X038 succinate esterExample 2.C X052 succinate ester Example 2.D X058 succinate esterExample 2.E X067 succinate ester Example 2.F X069 succinate esterExample 2.G General procedure for the high density loading of controlledpore glass supports with PAZ ligand succinates Example 2.H Synthesis ofX050, X059, X061, X062, X065, X068 alcohols and succinate esters Example2.I X060 and X064 alcohols and succinate esters Example 2.J. X063succinate ester Example 2.K X066 succinate ester Example 2.L X051succinate ester Example 2.M Synthesis of X097 succinate ester Example2.N Synthesis of X098 succinate ester Example 2.O Synthesis of siRNAconjugated with X109 Example 2.P Synthesis of siRNA conjugated with X110Example 2.Q Synthesis of siRNA conjugated with X111 Example 2.RSynthesis of siRNA conjugated with X112 Example 2.S Synthesis of siRNAconjugated with X113 Example 2.T General procedure for the high densityloading of controlled pore glass supports with PAZ ligand succinates

Example 2 The Synthesis of Various 3′ End Cap Succinate Esters andAlcohols are Presented Below

Example 2.A X027 succinate ester Example 2.B X038 succinate esterExample 2.C X052 succinate ester Example 2.D X058 succinate esterExample 2.E X067 succinate ester Example 2.F X069 succinate esterExample 2.G General procedure for the high density loading of controlledpore glass supports with PAZ ligand succinates Example 2.H Synthesis ofX050, X059, X061, X062, X065, X068 alcohols and succinate esters Example2.I X060 and X064 alcohols and succinate esters Example 2.J. X063succinate ester Example 2.K X066 succinate ester Example 2.L X051succinate ester Example 2.M Synthesis of X097 succinate ester Example2.N Synthesis of X098 succinate ester Example 2.O Synthesis of siRNAconjugated with X109 Example 2.P Synthesis of siRNA conjugated with X110Example 2.Q Synthesis of siRNA conjugated with X111 Example 2.RSynthesis of siRNA conjugated with X112 Example 2.S Synthesis of siRNAconjugated with X113 Example 2.T General procedure for the high densityloading of controlled pore glass supports with PAZ ligand succinates

2.A. Synthesis of X027 Succinate Ester

To a solution of compound 1 (10.0 g, 70.0 mmol) in DMF (200 mL) wereadded DMT-CI 2 (28.4 g, 84.0 mmol) and 2,6-lutidine (15.0 g, 140 mmol).The reaction mixture was stirred at rt overnight. The reaction mixturewas poured into ice water and extracted with EtOAc (3×500 mL). Theorganic extracts was dried over sodium sulfate and concentrated invacuum to give the crude product, which was purified by silica gelchromatography (heptane/ethyl acetate/NEt₃) to give the desired productas white solid (16 g, 36%). ¹H NMR (DMSO-d₆, 400 MHz): 3.73 (s, 6H),4.17 (s, 2H), 6.91 (d, J=8.8 Hz, 4H), 7.35-7.22 (m, 7H), 7.42 (d, J=7.6Hz, 2H), 7.48 (d, J=8.4 Hz, 1H), 7.83-7.80 (m, 1H), 8.33 (d, J=1.6 Hz,1H).

To a solution of compound 2 (8.0 g, 18 mmol) in dioxane (160 mL)/H₂O (40mL) were added 3-hydroxyphenylboronic acid 4 (3.5 g, 25 mmol), Pd(PPh₃)₄(1.1 g, 1.0 mmol), and Na₂CO₃ (4.0 g, 38 mmol). The reaction mixture wasbubbled with nitrogen gas and stirred at 90° C. overnight. Then reactionmixture was poured into water and extracted with EtOAc (3×800 mL). Theorganic extracts was dried over sodium sulfate, concentrated in vacuum,and purified by silica gel chromatography (heptane/ethyl acetate/NEt₃)to give 4 as an impure light yellow oil (6 g).

To a solution of compound 4 (10 g crude, 20 mmol) in acetone (600 mL)were added compound 5 (4.0 g, 17.6 mmol), K₂CO₃ (4.0 g, 28 mmol), and KI(316 mg, 1.9 mmol). The reaction mixture was stirred at refluxovernight. After the reaction mixture was cooled, the solvent wasconcentrated in vacuum. The reside was diluted with water and extractedwith EtOAc (3×800 mL). The organic phase was dried over sodium sulfateand concentrated in vacuum to give the crude product, which was purifiedby silica gel chromatography (heptane/ethyl acetate/NEt₃) to give 6 aslight yellow oil (9 g, 69%). ¹H NMR (DMSO-d₆, 400 MHz): 3.74 (s, 6H),3.84 (s, 3H), 4.19 (s, 2H), 6.93 (d, J=8.8 Hz, 4H), 7.11-7.08 (m, 1H),7.27-7.23 (t, J=7.2 Hz, 1H), 7.46-7.31 (m, 9H), 7.59-7.55 (t, J=7.6 Hz,1H), 7.68 (d, J=8.0 Hz, 1H), 7.79-7.75 (m, 2H), 7.84-7.80 (m, 1H),7.97-7.92 (m, 2H), 8.10 (s, 1H), 8.61 (d, J=1.6 Hz, 1H).

Lithium aluminum hydride (30.7 mL of 1.0 M suspension in THF, 30.7 mmol)was added to a solution of compound 6 (8.0 g, 12 mmol) in THF (150 mL)at 0° C. After 2 hours at 0° C., the reaction mixture was quenched withwater (200 mL), and then the reaction mixture was extracted withdichloromethane (3×200 mL), The combined organic phase was dried oversodium sulfate, filtered, and concentrated in vacuum to give the desiredproduct 7 as white solid (6.1 g, 80%). ¹H NMR (DMSO-d₆, 400 MHz): 3.74(s, 6H), 4.19 (s, 2H), 5.54 (d, J=5.6 Hz, 2H), 5.18 (s, 2H), 5.27-5.24(t, J=6.0 Hz, 1H), 6.93 (d, J=8.8 Hz, 4H), 7.10-7.07 (m, 1H), 7.47-7.23(m, 14H), 7.67 (d, J=8.0 Hz, 1H), 7.75 (s, 1H), 7.83-7.81 (m, 1H), 7.95(d, J=8.0 Hz, 1H), 8.61 (d, J=1.2 Hz, 1H).

To a solution of 2.00 g (3.21 mmol) 7 and 390 mg (3.21 mmol)N,N-dimethylaminopyridine (DMAP) in 10 mL dry pyridine under argon wasadded 640 mg (6.41 mmol) succinic anhydride (8). The reaction mixturewas stirred at room temperature for 17 h and then 0.5 mL water wasadded. Stirring was continued for 30 min. The reaction mixture wasdiluted with 100 mL dichloromethane and washed with 50 mL ice-cold 10%aqueous citric acid and water (2×50 mL). The aqueous layers werereextracted with 50 mL dichloromethane. The combined organic layers weredried over Na₂SO₄ and evaporated. The remaining oil was coevaporatedtwice with toluene and the crude product purified by silica gelchromatography (dichloromethane/methanol/triethylamine 97:2:1) to give1.35 g (1.64 mmol, 51%) 9 as an off-white foam. ¹H NMR (400 MHz, CDCl₃):1.12 (t, J=7.3 Hz, 9H), 2.51-2.55 (m, 2H), 2.59-2.62 (m, 2H), 2.89 (q,J=7.3 Hz, 6H), 3.72 (s, 6H), 4.17 (s, 2H), 5.08 (s, 4H), 5.68 (s br.,1H), 6.76-6.80 (m, 4H), 6.93 (dd, J=8.1, 2.5 Hz, 1H), 7.14-7.18 (m, 1H),7.21-7.34 (m, 10H), 7.41-7.46 (m, 4H), 7.62 (dd, J=5.1, 2.5 Hz, 2H),7.71 (dd, J=8.2, 1.9 Hz, 1H), 8.59 (d, J=1.5 Hz, 1H).

2. B Synthesis of X038 Succinate Ester

Into a 2000-mL 3-necked round-bottom flask was placed a solution of6-bromo-1H-indole-2-carboxylic acid 1 (100 g, 417 mmol) in methanol(1000 mL). This was followed by the addition of thionyl chloride (100 g,840 mmol) dropwise with stirring. The resulting solution was heated toreflux for 2 h. The reaction mixture was cooled to rt and a precipitatewas formed. The solids were collected by filtration, washing withmethanol, and dried in an oven under reduced pressure, giving 2 (95 g,90%) as a white solid.

Into a 2000-mL 3-necked round-bottom flask, purged and maintained withan inert atmosphere of nitrogen, was placed a solution of 2 (90 g, 354mmol) in ethylene glycol dimethyl ether (500 mL), water (500 mL),(pyridin-3-yl)boronic acid 3 (43.6 g, 355 mmol), NEt₃ (107 g, 1.06 mol),and Pd(PPh₃)₄ (9 g, 7.79 mmol). The resulting solution was heated toreflux overnight. The reaction mixture was cooled to room temperatureand was quenched by the addition of 800 mL of water, forming aprecipitate. The solids were collected by filtration, washing withwater, and dried in an oven under reduced pressure, giving 4 (78 g, 87%)as a brown solid.

Into a 2000-mL round-bottom flask was placed a solution of 4 (75 g, 297mmol) in DMF (500 mL). This was followed by the addition of NBS (53.5 g,301 mmol), portionwise. The resulting solution was stirred for 2 h atrt. The reaction was then quenched by the addition of 1000 mL of water,forming a precipitate. The solids were collected by filtration, washingwith water and dried in an oven under reduced pressure, giving 5 (70 g,71%) as a brown solid. ¹H NMR (400 MHz, CDCl₃): 3.94 (s, 3H), 7.51-7.58(m, 2H), 7.67-7.76 (m, 2H), 8.11 (d, J=7.6 Hz, 1H), 8.60 (d, J=4.4 Hz,1H), 8.91 (s, 1H), 12.48 (s, 1H).

Into a 2000-mL round-bottom flask was placed a solution of 5 (68 g, 205)in methanol (500 mL), water (100 mL), and sodium hydroxide (25 g, 625mmol). The resulting solution was heated to reflux for 2 hr. Theresulting solution was cooled to room temperature and diluted with 500mL of water. The pH value of the solution was adjusted to 5-6 with 2NHCl (aq), forming a precipitate. The solids were collected byfiltration, washing with water, and dried in an oven under reducedpressure, giving 6 (50 g, 77%) as a light yellow solid. ¹H NMR (400 MHz,CDCl₃): 7.50-7.53 (m, 2H), 7.65-7.71 (m, 2H), 8.09 (d, J=7.6 Hz, 1H),8.59 (d, J=4 Hz, 1H), 8.91 (s, 1H), 12.30 (s, 1H), 13.55 (s, 1H).

Into a 2000-mL round-bottom flask was placed a solution of2-aminoethan-1-ol 7a (30 g, 491 mmol) in THF (600 mL), Fmoc-OSu (166 g,491 mmol), and NEt₃ (199 g, 1.97 mol). The resulting solution wasstirred overnight at rt. The mixture was concentrated under vacuum andpurified by silica gel chromatography (ethyl acetate/petroleum ether),giving 7b (130 g, 93%) as a white solid.

Into a 2000-mL round-bottom flask was placed a solution of 7b (130 g,459 mmol) in pyridine (500 mL),1-[chloro(4-methoxyphenyl)benzyl]-4-methoxybenzene (DMT-CI) (233 g, 688mmol), and 4-dimethylaminopyridine (2.8 g, 22.9 mmol). The resultingsolution was stirred overnight at rt. The reaction was then quenched bythe addition of water, and the resulting solution was extracted withethyl acetate (3×500 mL). The combined organic layers were dried oversodium sulfate and concentrated in vacuum. The residue was purified bysilica gel chromatography (ethyl acetate/petroleum ether), giving 7c(210 g, 78%) as a brown solid.

Into a 2000-mL round-bottom flask was placed a solution of 7c (210 g,359 mmol) in dichloromethane (500 mL) and NEt₃ (500 mL). The resultingsolution was stirred overnight at rt. The resulting mixture wasconcentrated under vacuum. The residue was purified by silica gelchromatography (ethyl acetate/petroleum ether), giving 7 (95 g, 73%) abrown solid. ¹H NMR (400 MHz, CDCl₃) 2.42 (br. s, 2H), 3.70-3.82 (m,2H), 3.80 (s, 6H), 6.79-6.87 (m, 4H), 7.19-7.25 (m, 2H), 7.29 (d, J=9.2Hz, 2H), 7.33-7.40 (m, 3H), 7.49 (d, J=7.6 Hz, 2H).

Into a 2000-mL round-bottom flask was placed a solution of 6 (40 g, 126mmol) in DMF (800 mL), 7 (69 g, 190 mmol), HATU (96 g, 252 mmol), andi-Pr₂EtN (65 g, 503 mmol). The resulting solution was stirred for 4 h atrt and then quenched by the addition of 1000 mL of water. The resultingsolution was extracted with ethyl acetate (3×800 mL). The combinedorganic layers were dried over sodium sulfate and concentrated undervacuum. The residue was purified by silica gel chromatography (ethylacetate/petroleum ether), giving 8 (30 g, 36%) of as a light yellowsolid.

Into a 2000-mL round-bottom flask was placed a solution of4-(chloromethyl)benzoic acid 9a (50 g, 293 mmol) in THF (200 mL). Thiswas followed by the addition of 1 M BH₃/THF (586 mL, 586 mmol) dropwisewith stirring at 0° C. over 1 hr. The resulting solution was stirred for4 h at rt. The reaction was then quenched by the addition of 600 mL of 1N HCl. The solution was extracted with 500 ml of ethyl acetate. Theorganic layer was washed with 300 ml of sodium carbonate (aq.), and 300ml of brine. The organic layer was dried over sodium sulfate andconcentrated under vacuum giving 9b (35 g, 76%) as a white solid. ¹H NMR(400 MHz, CDCl₃) 4.50 (d, J=4.8 Hz, 2H), 4.75 (s, 2H), 5.21 (t, J=4.8Hz, 1H), 7.32 (d, J=7.6 Hz, 2H), 7.39 (d, J=7.6 Hz, 2H).

Into a 1000-mL 3-necked round-bottom flask was placed a solution 9b (35g, 223 mmol) in THF (300 mL) and TEA (68 g, 672 mmol). This was followedby the addition of TMS-CI (36.4 g, 335 mmol) dropwise with stirring. Theresulting solution was stirred overnight at rt. The reaction was thenquenched by the addition of 500 mL of water and extracted with 500 ml ofethyl acetate. The organic layer was washed with 500 ml of NaHCO₃ (aq.),500 ml of brine, and dried over sodium sulfate. The residue wasconcentrated under vacuum giving 9 (35 g, 68%) as colorless oil.

Into a 1000-mL 3-necked round-bottom flask was placed a solution of 8(30 g, 45.3 mmol) in DMF (300 mL), and sodium hydride (1.1 g, 45.8mmol). The mixture was stirred at rt for 0.5 h. A solution of 9 (15.5 g,67.8 mmol) in THF (100 ml) was then added, and the resulting solutionwas stirred overnight at 60° C. The reaction mixture was cooled to rtand quenched by the addition of 500 mL of water. The resulting solutionwas extracted with dichloromethane (3×500 mL), and the combined organiclayers were concentrated under vacuum. The residue was purified bysilica gel chromatography (ethyl acetate/petroleum ether), giving 10 (15g, 39%) as a white solid.

Into a 500-mL round-bottom flask was placed a solution of 10 (15 g, 17.6mmol) in THF (150 mL) and TBAF (7 g, 26.8 mmol). The resulting solutionwas stirred for 30 min at rt. The resulting solution was diluted with300 mL of water and extracted with 500 mL of ethyl acetate. The organiclayer was washed with water (2×300 mL) and 300 mL of brine, and driedover sodium sulfate. The resulting mixture was concentrated under vacuumand the crude product was re-crystallized from hexane, giving 11 (5.5 g,40%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃): 3.30 (m, 2H), 3.66-3.65(m, 2H), 3.78 (s, 6H), 4.51 (s, 2H), 5.68 (s, 2H), 6.84 (d, J=8.8 Hz,4H), 7.09 (d, J=8 Hz, 2H), 7.19-7.35 (m, 9H), 7.47-7.54 (m, 4H), 7.70(d, J=9.2 Hz, 2H), 8.10 (d, J=8 Hz, 1H), 8.51 (d, J=4.8 Hz, 1H), 8.79(s, 1H).

To a solution of 1.57 g (2.00 mmol) 11 and 244 mg (2.00 mmol)N,N-dimethylaminopyridine (DMAP) in 8 mL dry pyridine under argon wasadded 400 mg (4.00 mmol) succinic anhydride (12). The reaction mixturewas stirred at room temperature for 22 h and then 0.5 mL water wasadded. Stirring was continued for 30 min. The reaction mixture was takenup in 100 mL dichloromethane and washed with 50 mL ice-cold 10% aqueouscitric acid and water (2×50 mL). The aqueous layers were reextractedwith 50 mL dichloromethane. The combined organic layers were dried overNa₂SO₄ and evaporated. The remaining oil was coevaporated twice withtoluene and the crude product purified by silica gel chromatography(dichloromethane/methanol/triethylamine 94:5:1) to give 2.05 g (2.00mmol, quant.) 13 as an off-white foam. ¹H NMR (400 MHz, CDCl₃): 1.05 (t,J=7.2 Hz, 9H), 2.45 (t, J=6.5 Hz, 2H), 2.54 (t, J=6.5 Hz, 2H), 2.66 (q,J=7.2 Hz, 6H), 3.29 (t, J=4.9 Hz, 2H), 3.60 (q, J=5.1 Hz, 2H), 3.70 (s,6H), 4.97 (s, 2H), 5.75 (s, 2H), 6.72-6.76 (m, 4H), 7.01 (d, J=8.1 Hz,2H), 7.12-7.23 (m, 6H), 7.26-7.31 (m, 5H), 7.35-7.41 (m, 4H), 7.63 (d,J=8.3 Hz, 1H), 7.79 (dt, J=8.1, 1.9 Hz, 1H), 8.49 (dd, J=4.8, 1.5 Hz,1H), 8.73 (d, J=2.0 Hz, 1H).

2.C. Synthesis of X052 Succinate Ester

In a dried, two-neck roundbottom flask 3.33 g (17.8 mmol) 4-bromobenzylalcohol, 2.35 g (17.8 mmol) 4-ethynylbenzyl alcohol, 750 mg (1.07 mmol)bis-(triphenylphosphine)-palladiumdichloride, and 340 mg (1.78 mmol)copper(I)iodide were dissolved in 45 mL dry THF under argon. Then 12.4mL (9.21 g, 71.3 mmol) Hünig's base were added and the mixture heated toreflux for 4 h. The reaction mixture was cooled to rt, passed through apad of Hyflo, the filtercake washed with THF, and the filtrateevaporated to dryness. The crude product was purified by silica gelchromatography (dichloromethane/methanol 99:1 to 49:1) to give 3 (1.03g, 24%) as a yellowish solid. ¹H NMR (400 MHz, DMSO-d₆): 4.54 (d, J=5.6Hz, 4H), 5.29 (t, J=5.8 Hz, 2H), 7.37 (d, J=8.3 Hz, 4H), 7.51 (d, J=8.1Hz, 4H).

Diol 3 (960 mg, 4.03 mmol) was dissolved in 17 mL pyridine under argonand cooled to 0° C. Then 4,4′-dimethoxytriphenylchloromethane (DMT-CI,1.37 mg, 4.03 mmol) was added portionwise over 15 min. The solution wasstirred overnight at ambient temperature. The reaction mixture wasdissolved in 100 mL dichloromethane and extracted twice with 50 mL sat.aqueous NaHCO₃ each. The aqueous layers where reextracted with 100 mLdichloromethane. The combined organic layers were dried over Na₂SO₄ andevaporated to dryness. The crude product was coevaporated twice withtoluene and purified by silica gel chromatography (heptane/ethyl acetate3:1 to 2:1 with 0.1% Et₃N) to give 4 as a foam in 61% yield (1.32 g,2.44 mmol). ¹H NMR (400 MHz, CDCl₃): 1.67 (t br., 1H), 3.72 (s, 6H),4.11 (s, 2H), 4.64 (s br., 2H) 6.76-6.79 (m, 4H), 7.13-7.17 (m, 1H),7.21-7.34 (m, 10H), 7.41-7.47 (m, 6H).

To a solution of 1.30 g (2.40 mmol) 4 and 290 mg (2.40 mmol)N,N-dimethylaminopyridine (DMAP) in 12 mL dry pyridine under argon wasadded 480 mg (4.81 mmol) succinic anhydride (5). The reaction mixturewas stirred at room temperature for 19 h and then quenched by additionof 1.5 mL water. Stirring was continued for 60 min before the reactionmixture was diluted with 150 mL dichloromethane and washed with 75 mLice-cold 10% aqueous citric acid and water (2×75 mL). The aqueous layerswere reextracted with 150 mL dichloromethane. The combined organiclayers were dried over Na₂SO₄ and evaporated. The remaining oil wascoevaporated twice with toluene and the crude product purified by silicagel chromatography (dichloromethane/methanol/triethylamine 97:2:1) togive 1.36 g (1.83 mmol, 76%) 6 as an off-white, sticky foam. ¹H NMR (400MHz, CDCl₃): 1.16 (t, J=7.3 Hz, 9H), 2.50 (t, J=6.5 Hz, 2H), 2.60 (t,J=6.7 Hz, 2H), 2.87 (q, J=7.3 Hz, 6H), 3.72 (s, 6H), 4.11 (s, 2H), 5.05(s, 2H), 5.65 (s br., 1H), 6.75-6.79 (m, 4H), 7.13-7.16 (m, 1H),7.21-7.34 (m, 10H), 7.41-7.44 (m, 6H).

2.D. Synthesis of X058 Succinate Ester

A 1M solution of boron trichloride in toluene (4.94 L, 4.94 mol) underargon was diluted with 5 L of toluene before a solution of 850 g (4.94mol) 2-bromoaniline (1) in 400 mL toluene was added over a period of 40min, resulting in a clear pale brown solution in a slightly exothermicreaction. The temperature was kept below 24° C. with cooling. Then asolution of 1529 g (14.8 mol) benzonitrile (2) in 1.53 L toluene wasadded, followed by 725 g (5.44 mol) AlCl₃ resulting in a fine suspensionwhich turned pale green. This mixture was stirred for 1 h at 20° C. to24° C., then heated to reflux for 6 h. After 1 h at reflux a clear palebrown solution resulted, which changed to pale yellow after 4 h andeventually became turbid. After a total of 7 h, the reaction mixture wasallowed to cool to 20° C. overnight, resulting in an emulsion, and wasthen quenched by addition of 15 L ice cold 1M HCl (caution: exothermicreaction with strong gas evolution at the beginning of the addition).The temperature was kept at 22° C. to 35° C. by cooling. This biphasicmixture was warmed to 80° C. for 60 minutes. The aqueous phase wasseparated and reextracted with 5 L of toluene. Both organic phases werewashed with 5 L 1M HCl, 10 L of a 2M NaOH solution and 5 L brine. Thecombined toluene layers were dried over MgSO₄ and evaporated (55° C., 5mbar) to a brown oil which was further dried at 80° C. and 0.5 mbar. Thecrude product solidified after 1 h at room temperature and was thenpurified by silica gel chromatography (heptane/ethyl acetate) yielding812 g (2.94 mol, 58%) 3. ¹H NMR (400 MHz, acetonitrile-d₃): 6.52-6.68(m, 3H), 7.42 (dd, J=7.8, 1.3 Hz, 1H), 7.47-7.54 (m, 2H), 7.57-7.64 (m,3H), 7.66 (dd, J=7.8, 1.3 Hz, 1H).

Under argon and with vigorous stirring 120 g (1033 mmol)methyl-4-oxobutanoate (4) was added at once to a solution ofbenzophenone 3 (233 g, 808 mmol) in 3.5 L glacial acid, resulting in aclear yellow solution. After addition of 2.5 mL (4.60 g, 46.9 mmol)concentrated sulfuric acid the color changed to pale red. The solutionwas heated to reflux overnight. The yellow solution was then cooled toroom temperature and slowly poured in to an ice cold solution of 3 kgammonium chloride in 10 L of water. The mixture was extracted twice with5 L of dichloromethane each. The combined organic layers were extractedtwice with 6 L saturated, aqueous NaHCO₃ solution (caution: gasevolution). The organic layer was dried over MgSO₄ and evaporated todryness to give 338 g of crude product as pale yellow solid. Thismaterial was crystallized from 6 L heptane/ethyl acetate 4:1 yielding136 g (382 mmol, 47%) 5 as colorless crystals. ¹H NMR (400 MHz,acetonitrile-d₃): 3.58 (s, 3H), 3.65 (s, 2H), 7.25-7.31 (m, 2H),7.32-7.38 (m, 1H), 7.40-7.45 (m, 1H), 7.53-7.61 (m, 3H), 8.07 (dd,J=7.6, 1.5 Hz, 1H), 8.97 (s, 1H).

Phenylquinoline 5 (338 g, 949 mmol), vinyl boronate 6 (175 g, 1139mmol), and potassium carbonate (266 g, 1926 mmol) were dissolved in 4.6L 1,4-dioxane/water 1:1 under argon. The mixture was stirred for 5 minbefore adding 31.9 g (78 mmol)S—PHOS and 10.0 g (44.6 mmol)palladium(II)acetate. The mixture was warmed to 70° C. and stirred underargon for 5 h. The yellow mixture was then cooled to room temperature,diluted with 3 L tert.-butylmethylether and extracted twice with 2.5 Lwater, followed by 2 L brine. The aqueous phases were reextracted with 2L tert.-butylmethylether. The combined organic layers were dried withMgSO₄ and evaporated to dryness resulting in 348 g of a yellow oil. Thecrude product was purified by silica gel chromatography (heptane/ethylacetate 4:1) giving 196 g (645 mmol, 68%) 7. ¹H NMR (400 MHz,acetonitrile-d₃): 3.58 (s, 3H), 3.63 (s, 2H), 5.50 (dd, J=11.1, 1.5 Hz,1H), 6.04 (dd, J=17.9, 1.8 Hz, 1H), 7.24-7.30 (m, 2H), 7.35 (dd, J=8.3,1.3 Hz, 1H), 7.43-7.50 (m, 1H), 7.51-7.59 (m, 3H), 7.97 (dd, J=7.1, 1.0Hz, 1H), 8.06 (dd, J=17.9, 11.4 Hz, 1H), 8.91 (s, 1H)

Vinyl-quinoline 7 (194 g, 640 mmol) was dissolved in 3 L THF underargon. The yellow solution was cooled to 15° C. and stirred for 10 min.Then 1.8 L of a 0.5M solution of 9-borabicylo[3.3.1]nonane in THF (900mmol) was added dropwise during a period of 30 min at 15 to 18° C.Stirring was continued at room temperature overnight. After cooling to−50° C. (dry ice/acetone), 300 mL of a 30% hydrogen peroxide solution inwater (2937 mmol) was added dropwise over 5 min (exothermic reaction),followed by the addition of 520 mL of a 3M aqueous NaOH solution (1560mmol) which resulted in a yellow suspension. The reaction mixture wasallowed to warm to 0 to 2° C. and then stirred for 3 h at thistemperature. The yellow suspension was diluted with 3 L water and thenextracted twice with 3 L ethyl acetate. Both organic layers were washedwith 2 L water followed by 2 L brine. The combined organic phases weredried over MgSO₄ and evaporated to give a pale brown oil which waspurified by silica gel chromatography (2-3% methanol in dichloromethane)yielding 163 g (507 mmol, 79%) 8. ¹H NMR (400 MHz, DMSO-d₆): 3.42 (t,J=6.8 Hz, 2H), 3.53 (s, 3H), 3.65 (s, 2H), 3.76-3.84 (m, 2H), 4.54 (t,J=5.3 Hz, 1H), 7.19 (dd, J=8.6, 1.5 Hz, 1H), 7.21-7.25 (m, 2H), 7.40(dd, J=8.1, 7.1 Hz, 1H), 7.49-7.59 (m, 3H), 7.62 (d, J=7.1 Hz, 1H), 8.91(s, 1H).

Methyl ester 8 (64.5 g, 201 mmol) was dissolved in 600 ml methanol. Tothis solution was added 450 mL of 0.5M aqueous NaOH (225 mmol). Theturbid solution was stirred for 1 h at 50° C. Then the reaction mixturewas evaporated to about 600 mL and the residue extracted twice with 800mL tert.-butylmethylether each. The ether layers were washed with 300 mLwater. The combined water phases were evaporated to dryness and theresidue coevaporated twice with toluene to give 67 g of a beige solid.This material was dissolved in 1 L water and then 250 mL 1M aqueouscitric acid was added carefully. The resulting suspension was stirredfor 15 min and then extracted twice with 1 L ethyl acetate each. Theorganic layers were dried over MgSO4 and evaporated to dryness, yielding54.1 g (176 mmol, 88%) acid 9 as beige solid. ¹H NMR (400 MHz, D₂O):3.80 (t, J=6.8 Hz, 2H), 3.82 (s, 2H), 4.32 (t, J=6.8 Hz, 2H), 7.58-7.64(m, 2H), 7.67-7.73 (m, 1H), 7.73-7.79 (m, 1H), 7.87-7.95 (m, 3H), 7.97(dd, J=7.1, 1.5 Hz, 1H), 9.14 (s, 1H).

Phthalic anhydride (10B, 140 g, 945 mmol) was mixed with4-amino-1-butanol (10A) and heated to 140° C. for 3 hours. Over thecourse of the reaction, the colorless suspension turned into clear,light yellow liquid. The mixture was allowed to cool to 80° C. andpoured onto 3 kg of crushed ice. The ice mixture was extracted threetimes with 2 L of dichloromethane each. The combined organic phases werewashed with 2 L saturated aqueous NaHCO₃, twice with 2 L water, and thenwith 2 L brine. The organic layer was dried over MgSO₄ and concentratedto give 195 g 10C (889 mmol, 95%) as beige solid This material was usedin the next step without further purification. ¹H NMR (400 MHz,acetonitrile-d₃): 1.48-1.58 (m, 2H), 1.67-1.78 (m, 2H), 2.37 (t, J=5.3Hz, 1H), 3.50-3.57 (m, 2H), 3.66 (t, J=7.3 Hz, 2H), 7.75-7.85 (m, 4H).

Phthalimide 10C (193 g, 880 mmol) was dissolved in 2.5 L pyridine underargon. Then 4,4′-dimethoxytriphenylchloromethane (DMT-CI, 328 g, 968mmol) was added in four portions over 10 min. The temperature of thereaction mixture rose from 23° C. to 26° C. and the yellow solutionturned red, then back to yellow again. The solution was stirredovernight at ambient temperature. To quench the reaction 200 mL methanolwas added 200 ml and the reaction mixture subsequently evaporated. Theresidue was dissolved in 5 L ethyl acetate and extracted twice with 5 L5% aqueous citric acid, once with 5% aqueous NaHCO₃ and finally with 5 Lbrine. The aqueous layers where reextracted with 2 L ethyl acetate. Thecombined organic layers were dried over MgSO₄ and evaporated to dryness.The crude product, 495 g of a brown oil, was purified by silica gelchromatography (heptane/ethyl acetate 4:1 to 3:1). DMT-protected linker10D was obtained in 81% yield (381 g, 730 mmol). ¹H NMR (400 MHz,DMSO-d₆): 1.48-1.60 (m, 2H), 1.62-1.74 (m, 2H), 3.01 (t, J=6.1 Hz, 2H),3.56 (t, J=7.1 Hz, 2H), 3.73 (s, 6H), 6.82-6.88 (m, 4H), 7.16-7.25 (m,5H), 7.25-7.31 (m, 2H), 7.32-7.37 (m, 2H), 7.78-7.87 (m, 4H).

Phthalimide 10D (302 g, 579 mmol) was dissolved in 7 L ethanol at 50° C.and 320 mL (327 g, 3.57 mol) hydrazine hydrate was added. The reactionmixture was heated for 5 h to 50° C. The colorless suspension was cooledto room temperature and diluted with 15 L of water. The resultingemulsion was extracted twice with 6 L tert.-butylmethylether each. Theorganic phases were washed twice with 4 L 5% aqueous NaHCO₃, then with 4L brine. The combined ether layers were dried over MgSO₄ and evaporatedto give 226 g (578 mmol) 10 as a pale yellow oil which was used in thenext step without additional purification. ¹H NMR (400 MHz,acetonitrile-d₃): 1.42-1.54 (m, 2H), 1.56-1.66 (m, 2H), 2.61 (t, J=7.1Hz, 2H), 3.06 (t, J=6.6 Hz, 2H), 3.78 (s, 6H), 6.84-6.90 (m, 4H),7.19-7.26 (m, 1H), 7.28-7.35 (m, 6H), 7.41-7.47 (m, 2H).

Quinoline acetic acid 9 (92 g, 279 mmol) was dissolved in 1.5 L DMFunder argon and a solution of 128 g (327 mmol) DMT-protectedaminobutanol 10 in 1 L DMF followed by 146 mL (108 g, 838 mmol)ethyldiisopropylamine were added. Finally, 138 g (363 mmol) HATU wasadded to the pale yellow, turbid solution, resulting in an exothermicreaction. The temperature was kept below 25° C. with ice-bath cooling.The reaction mixture was stirred at room temperature for 6 h and thendiluted with 3 L aqueous NaHCO₃. This mixture was extracted twice with 3L tert.-butylmethylether each. The organic layers were washed withbrine, combined, dried, and evaporated. The crude product (180 g palebrown oil) was purified by silica gel chromatography(dichloromethane/methanol/triethylamine 98:2:0.25) to give 134 g (197mmol, 70%) 11 as colorless foam. ¹H NMR (400 MHz, DMSO-d₆): 1.35-1.57(m, 4H), 2.97 (t, J=6.3 Hz, 4H), 3.34-3.48 (m, 4H), 3.73 (s, 6H),3.76-3.83 (m, 2H), 4.54 (t, J=5.3 Hz, 1H), 6.84-6.90 (m, 4H), 7.15-7.32(m, 10H), 7.34-7.41 (m, 3H), 7.42-7.53 (m, 3H), 7.58 (dd, J=6.8, 1.3 Hz,1H), 7.62 (t, J=4.8 Hz, 1H), 8.83 (s, 1H).

Alcohol 11 (43.8 g, 64.4 mmol) and N,N-dimethylaminopyridine (DMAP, 7.87g, 64.4 mmol) were dissolved in 600 mL pyridine under argon. Then 12.9 g(128 mmol) succinic anhydride (12) was added and the reaction mixturestirred at room temperature for 20 h. The reaction was quenched byaddition of 10 mL water and stirring continued for 30 min. The reactionmixture was diluted with 1200 mL dichloromethane and washed with 600 mLice-cold 10% aqueous citric acid and twice with 600 mL water. Theaqueous layers were reextracted with 600 mL dichloromethane. Thecombined organic layers were dried over Na₂SO₄ and evaporated. The crudeproduct was coevaporated twice with 100 mL toluene and then purified bysilica gel chromatography (dichloromethane/methanol/triethylamine 97:2:1to 94:5:1) to give 57.5 g (quantitative) 13 as an off-white foam. ¹H NMR(400 MHz, CDCl₃): 1.17 (t, J=7.3 Hz, 9H), 1.46-1.60 (m, 4H), 2.52 (t,J=7.2 Hz, 2H), 2.61 (t, J=7.0 Hz, 2H), 2.82 (q, J=7.3 Hz, 6H), 3.06 (t,J=5.8 Hz, 2H), 3.16 (q, J=6.3 Hz, 2H), 3.49 (s, 2H), 3.64 (t, J=6.8 Hz,2H), 3.80 (s, 6H), 4.53 (t, J=7.5 Hz, 2H), 5.38 (t br., 1H), 6.08 (sbr., 1H), 6.80-6.84 (m, 4H), 7.20 (t, J=7.3 Hz, 1H), 7.26-7.38 (m, 10H),7.41-7.52 (m, 5H), 7.59 (d, J=6.3 Hz, 1H), 8.92 (s, 1H).

2.E. Synthesis of X067 Succinate Ester

To a 250 mL roundbottom flask was added 2-(4-bromophenyl)ethanol 1 (1.00g, 4.97 mmol), pyridine (25 mL) and4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (DMT-CI) (1.69 g, 4.97mmol). The solution was stirred at rt for 2 h. 1 mL of MeOH was added,and the solution was stirred at rt for 10 min. The solution was thenconcentrated under vacuum, dissolved in 250 mL of EtOAc, and washed with100 mL sat. aq. NaHCO₃, 100 mL of water, and 100 mL of brine. Theorganic layer was dried with sodium sulfate, concentrated under vacuum,and purified by silica gel chromatography (heptane/ethyl acetate/NEt₃)to give 2 (2.35 g, 94%) as a foamy solid. ¹H NMR (400 MHz, DMSO-d₆):2.80 (t, J=6.6 Hz, 2H), 3.12 (t, J=6.6 Hz, 2H), 3.72 (s, 6H), 6.81-6.87(m, 4H), 7.12-7.22 (m, 7H), 7.26 (d, J=4.0 Hz, 4H), 7.44-7.50 (m, 2H).

To a 40 mL glass vial with rubber septa was added 2 (0.70 g, 1.39 mmol),4-(hydroxymethyl)phenylboronic acid 3 (0.25 g, 1.67 mmol), Pd(PPh₃)₄ (80mg, 0.070 mmol), 2 M (aq) Na₂CO₃ (2.1 mL, 4.17 mmol), and 1,4-dioxane (7mL). The contents were briefly placed under vacuum, and then placedunder a nitrogen atmosphere. The vial was sealed and heated at 90° C.for 16 h. After cooling to rt, EtOAc was added and the mixture waswashed with sat. aq. NaHCO₃ and brine. The organic portion was driedwith sodium sulfate, concentrated under vacuum, and purified by silicagel chromatography (heptane/ethyl acetate/NEt₃) to give 3 (0.64 g, 87%)as a foamy solid. ¹H NMR (400 MHz, DMSO-d₆): 2.86 (t, J=6.6 Hz, 2H),3.16 (t, J=6.6 Hz, 2H), 3.72 (s, 6H), 4.52 (d, J=6.1 Hz, 2H), 5.19 (t,J=5.8 Hz, 1H), 6.81-6.87 (m, 4H), 7.18 (d, J=9.1 Hz, 4H), 7.20-7.22 (m,1H), 7.24-7.33 (m, 6H), 7.38 (d, J=8.6 Hz, 2H), 7.57 (d, J=8.1 Hz, 2H),7.60 (d, J=8.1 Hz, 2H).

To a solution of 3.68 g (6.93 mmol) 4 and 847 mg (6.93 mmol)N,N-dimethylaminopyridine (DMAP) in 35 mL dry pyridine under argon wasadded 1.39 g (13.9 mmol) succinic anhydride (5). The reaction mixturewas stirred at room temperature for 16 h and then 2.5 mL water wasadded. Stirring was continued for 30 min. The reaction mixture was takenup in 300 mL dichloromethane and washed with 150 mL ice-cold 10% aqueouscitric acid and water (2×150 mL). The aqueous layers were reextractedwith 150 mL dichloromethane. The combined organic layers were dried overNa₂SO₄ and evaporated. The remaining oil was coevaporated twice withtoluene and the crude product purified by silica gel chromatography(dichloromethane/methanol/triethylamine 97:2:1) to give 4.68 g (6.39mmol, 92%) 6 as an off-white foam. ¹H NMR (400 MHz, CDCl₃): 1.14 (t,J=7.4 Hz, 9H), 2.51 (t, J=6.8 Hz, 2H), 2.60 (t, J=6.5 Hz, 2H), 2.82-2.90(m, 8H), 3.24 (t, J=6.8 Hz, 2H), 3.70 (s, 6H), 5.08 (s, 2H), 6.69-6.73(m, 4H), 7.08-7.21 (m, 9H), 7.28-7.35 (m, 4H), 7.42 (d, J=8.1 Hz, 2H),7.48 (d, J=8.0 Hz, 2H), 8.04 (s br., 1H).

2. F. Synthesis of X069 Succinate Ester

Compound 2 was prepared according to Eur. J. Org. Chem, 2002, 19,3326-3335. In a 250 mL roundbottom was added 3-bromobenzaldehyde 1 (10.0g, 54.0 mmol) and EtOH (25 mL). The solution was cooled to 0° C. in anice-water bath, sodium borohydride (1.11 g, 29.5 mmol) was added, andthe mixture was stirred at 0° C. for 1 h. Sodium sulfate decahydrate wasadded, and the reaction was stirred at rt for 1 h to quench theborohydride. Diethyl ether was added, and the mixture was washed withwater. The organic layer was dried with sodium sulfate and concentratedunder vacuum to give 2 (9.50 g, 94%) as a clear oil. ¹H NMR (400 MHz,CDCl₃): 2.34 (br. s, 1H), 4.61 (br. s, 2H), 7.13-7.33 (m, 2H), 7.40 (d,J=7.6 Hz, 1H), 7.49 (s, 1H).

To a 500 mL roundbottom flask was added 2 (9.50 g, 50.8 mmol),4,4′-(chloro(phenyl)methylene)bis(methoxybenzene) (DMT-CI) (17.2 g, 50.8mmol), DMAP (0.310 g, 0.050 mmol), and pyridine (200 mL). The solutionwas placed under an atmosphere of nitrogen, and stirred overnight at rt.EtOAc was added, and the solution was washed with sat. aq. NaHCO₃. Theorganic layer was concentrated under vacuum, and purified by silica gelchromatography (heptane/ethyl acetate/NEt₃) to give 3 (23.3 g, 94%) as afoamy solid. ¹H NMR (400 MHz, CDCl₃): 3.74 (s, 6H), 4.12 (s, 2H), 6.92(dd, J=8.0 Hz, 4H), 7.21-7.48 (m, 13H).

To a 40 mL glass vial with rubber septum was added 3 (1.00 g, 2.04mmol), 4-ethynylbenzyl alcohol 4 (0.405 g, 3.06 mmol), PdCl₂(PPh₃)₂ (86mg, 0.123 mmol), CuI (39 mg, 0.204 mmol), iPr₂EtN (1.06 g, 8.17 mmol)and THF (7 mL). The contents were briefly placed under vacuum, and thenplaced under a nitrogen atmosphere. The vial was sealed and heated at70° C. for 16 h. After cooling to rt, the mixture was filtered throughcelite, washing with EtOAc, and the filtrated was concentrated undervacuum. The residue was purified by silica gel chromatography(heptane/ethyl acetate/NEt₃) to give 5 (0.680 g, 62%) as a foamy solid.¹H NMR (400 MHz, CDCl₃): 3.74 (s, 6H), 4.12 (s, 2H), 4.53 (d, J=4.0 Hz,2H), 5.29 (t, J=4.0 Hz, 1H), 6.93 (dd, J=8.0 Hz, 4H), 7.19-7.58 (m,17H).

To a solution of 4.55 g (8.42 mmol) 5 and 1.03 g (8.42 mmol)N,N-dimethylaminopyridine (DMAP) in 42 mL dry pyridine under argon wasadded 1.68 g (16.8 mmol) succinic anhydride (6). The reaction mixturewas stirred at room temperature for 16 h and then 2.5 mL water wasadded. Stirring was continued for 30 min. The reaction mixture was takenup in 300 mL dichloromethane and washed with 150 mL ice-cold 10% aqueouscitric acid and water (2×150 mL). The aqueous layers were reextractedwith 150 mL dichloromethane. The combined organic layers were dried overNa₂SO₄ and evaporated. The remaining oil was coevaporated twice withtoluene and the crude product purified by silica gel chromatography(dichloromethane/methanol/triethylamine 97:2:1) to give 5.81 g (7.83mmol, 93%) 7 as an off-white, sticky foam. ¹H NMR (400 MHz, CDCl₃): 1.14(t, J=7.4 Hz, 9H), 2.50 (t, J=6.5 Hz, 2H), 2.60 (t, J=6.6 Hz, 2H), 2.86(q, J=7.3 Hz, 6H), 3.72 (s, 6H), 4.09 (s, 2H), 5.05 (s, 2H), 5.95 (sbr., 1H), 6.75-6.79 (m, 4H), 7.15 (tt, J=7.3, 1.5 Hz, 1H), 7.21-7.36 (m,11H), 7.42-7.45 (m, 5H).

2.G. General Procedure for the High Density Loading of Controlled PoreGlass Supports with PAZ Ligand Succinates

In an Erlenmeyer flask 1.00 mmol PAZ ligand succinate salt 1 wasdissolved in 50 mL dry acetonitrile under argon. To this solution 353 mg(1.10 mmol)O-(1H-benzo-1,2,3-triazol-1-yl)-N,N,N′,N′-tetramethyl-uroniumtetrafluoroborate (TBTU) was added and the solution shaken for 10 min.Then 10 g long chain alkylamine controlled pore glass (LCAA/CNA-600-CPG,PrimeSynthesis, 2) was added and the reaction mixture gently agitatedfor 5 min. Finally, 0.685 mL (517 mg, 4.00 mmol) Hünig's base was addedand the flask gently shaken for 24 h on an orbital shaker. Loadingdensity was assessed by detritylating an aliquote of the CPG (3-5 mg CPGwashed with acetonitrile, dried in vacuo, added to 25 mL 3%dichloroacetic acid in dichloromethane (v/v), absorbance at 504 nmdetermined). If loading density was in the desired range (60-90micromol/g), the CPG was filtered off and washed extensively withacetonitrile. Underivatized amino groups were capped by treating the CPGwith x mL each of a mixture of acetic anhydride/2,6-lutidine/THF 1:1:8(v/v/v) and a solution of 1-methylimidazole in THF 16:84 (v/v). Themixture was gently shaken for 15 min at room temperature. Then the CPGwas filtered off, washed with acetonitrile and dried under vacuumovernight. Loading density was determined again as above. Loading yieldsfor the succinates in examples 1-6 were in the range of 64-75micromol/g.

2.H. Synthesis of X050, X059, X061, X062, X065, X068 Alcohols andSuccinate Esters Prepared in an Analogous Manner to X027

X050 alcohol: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.73 (s, 6H) 4.19 (s, 2H)4.51 (d, J=5.56 Hz, 2H) 5.17 (s, 2H) 5.21 (t, J=5.81 Hz, 1H) 6.89-6.95(m, 4H) 7.01 (dd, J=8.08, 2.02 Hz, 1H) 7.19-7.30 (m, 4H) 7.30-7.40 (m,9H) 7.40-7.49 (m, 5H) 7.53 (s, 1H) 7.57 (d, J=7.58 Hz, 1H). MS (ESI−)m/z: calcd for C₄₂H₃₈O₅622.3. found 667.9 [MH⁻+formic acid].

X059 alcohol: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.74 (s, 6H) 4.10 (s, 2H)4.52 (s, 2H) 5.15 (s, 2H) 5.22 (br. s., 1H) 6.90-6.96 (m, 4H) 7.07-7.13(m, 2H) 7.22-7.30 (m, 2H) 7.30-7.38 (m, 8H) 7.39-7.48 (m, 5H) 7.60 (d,J=8.08 Hz, 4H). MS (ESI−) m/z: calcd for C₄₂H₃₈O₅ 622.3. found 621.1[MH⁻].

X061 alcohol: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.74 (s, 6H) 4.17 (s, 2H)4.63 (d, J=5.05 Hz, 2H) 5.17-5.22 (m, 3H) 6.93 (d, J=8.59 Hz, 4H) 7.11(d, J=8.59 Hz, 2H) 7.22-7.37 (m, 10H) 7.40-7.52 (m, 7H) 7.58 (d, J=8.59Hz, 2H). MS (ESI−) m/z: calcd for C₄₂H₃₈O₅622.3. found 667.6 [MH⁻+formicacid].

X062 alcohol: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.74 (s, 6H) 4.16 (s, 2H)4.52 (d, J=6.06 Hz, 2H) 5.14 (s, 2H) 5.19-5.23 (m, 1H) 6.90-6.95 (m, 4H)7.07-7.12 (m, 2H) 7.21-7.29 (m, 2H) 7.30-7.38 (m, 9H) 7.39-7.48 (m, 5H)7.49-7.53 (m, 1H) 7.55-7.60 (m, 2H). MS (ESI−) m/z: calcd forC₄₂H₃₈O₅622.3. found 667.7 [MH⁻+formic acid].

X065 alcohol: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.75 (s, 6H) 4.16 (s, 2H)4.52 (d, J=6.06 Hz, 2H) 5.17 (s, 2H) 5.21 (t, J=5.81 Hz, 1H) 6.93 (d,J=8.59 Hz, 4H) 7.12 (d, J=9.09 Hz, 2H) 7.22-7.39 (m, 10H) 7.41-7.47 (m,3H) 7.78 (dd, J=8.34, 2.27 Hz, 1H) 7.85-7.90 (m, 1H) 8.03 (d, J=9.09 Hz,2H) 8.55 (d, J=1.52 Hz, 1H). MS (ESI+) m/z: calcd for C₄₁H₃₇NO₅ 623.3.found 624.7 [MH⁺].

X068 alcohol: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.87 (t, J=6.57 Hz, 2H)3.16 (t, J=6.57 Hz, 2H) 3.72 (s, 6H) 4.51 (d, J=5.56 Hz, 2H) 5.17 (s,2H) 5.21 (t, J=5.81 Hz, 1H) 6.85 (d, J=8.59 Hz, 4H) 6.99 (dd, J=8.08,1.52 Hz, 1H) 7.18 (d, J=9.09 Hz, 4H) 7.20-7.38 (m, 13H) 7.43 (s, 1H)7.59 (d, J=8.59 Hz, 2H). MS (ESI+) m/z: calcd for C₄₃H₄₀O₅636.3. found659.7 [M+Na].

2.I. Synthesis of X060 and X064 Alcohols and Succinate Esters

Prepared in an analogous manner to X067

X060 alcohol: ¹H NMR (400 MHz, DMSO-d₆) δ ppm ¹H NMR (400 MHz, DMSO-d₆)δ ppm 3.75 (s, 6H) 4.12 (s, 2H) 4.57 (d, J=5.56 Hz, 2H) 5.20-5.26 (m,1H) 6.90-6.96 (m, 4H) 7.22-7.28 (m, 1H) 7.29-7.38 (m, 7H) 7.39-7.48 (m,5H) 7.53 (d, J=8.08 Hz, 1H) 7.60 (s, 1H) 7.64 (d, J=8.08 Hz, 2H). MS(ESI+) m/z: calcd for C₃₅H₃₂O₄516.2. found 303.4 [DMT⁺].

X064 alcohol: ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.75 (s, 6H) 4.18 (s, 2H)4.56 (d, J=5.56 Hz, 2H) 5.24 (t, J=5.81 Hz, 1H) 6.93 (d, J=9.09 Hz, 4H)7.25 (t, J=7.33 Hz, 1H) 7.32 (d, J=9.09 Hz, 4H) 7.34-7.39 (m, 2H) 7.44(t, J=8.08 Hz, 4H) 7.82 (dd, J=8.34, 2.27 Hz, 1H) 7.93 (d, J=8.08 Hz,1H) 8.04 (d, J=8.08 Hz, 2H) 8.59 (d, J=2.02 Hz, 1H). MS (ESI+) m/z:calcd for C₃₄H₃₁NO₄ 517.2. found 518.8 [MH⁺].

2.J. Synthesis of X063 Succinate Ester

3-(hydroxymethyl)phenol (1, 6.21 g, 50.0 mmol) was dissolved in pyridine(100 mL) and cooled to 0° C. DMT-CI (16.9 g, 50 mmol) was added and thesolution was stirred at rt for 2 h. 500 mL of EtOAc was added, thesolution was washed 1× each with 400 mL sat. aq. NaHCO₃, water, andbrine. The organic portion was dried with Na₂SO₄, filtered, andconcentrated under vacuum. The mixture was re-dissolved inacetone/toluene and concentrated, repeating this process 4-times. Theresidue was then concentrated under vacuum overnight to give 2 (20.9 g,98%) as a foamy solid.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.74 (s, 6H) 3.97 (s, 2H) 6.66 (dd,J=8.08, 1.52 Hz, 1H) 6.72 (d, J=7.58 Hz, 1H) 6.83 (d, J=1.52 Hz, 1H)6.89-6.95 (m, 4H) 7.12 (t, J=7.83 Hz, 1H) 7.17 (d, J=7.58 Hz, 1H)7.27-7.32 (m, 4H) 7.34 (t, J=7.58 Hz, 2H) 7.40-7.46 (m, 2H) 9.37 (s,1H).

To compound 2 (17.2 g, 36.3 mmol) in acetone (145 mL) was added methyl4-bromo-3-(bromomethyl)benzoate (3, 11.7 g, 38.1 mmol) and K₂CO₃ (30.1g, 218 mmol). The flask was evacuated/N₂ backfilled 2×, and heated atreflux overnight under an atmosphere of N₂. After cooling to rt, themixture was filtered, washing with CH₂Cl₂, and concentrated. The residuewas then redissolved in CH₂Cl₂, dried with Na₂SO₄, filtered, andconcentrated. To give 4 (24.8 g, 99%) as a foamy solid. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 3.74 (s, 6H) 3.84 (s, 3H) 4.07 (s, 2H) 5.20 (s, 2H)6.88-6.92 (m, 4H) 6.94-6.98 (m, 2H) 6.99 (d, J=1.52 Hz, 1H) 7.21-7.26(m, 1H) 7.26-7.30 (m, 5H) 7.30-7.35 (m, 2H) 7.38-7.43 (m, 2H) 7.85 (s,2H) 8.11 (s, 1H)

To compound 4 (24.8 g, 35.8 mmol) in dimethoxyethane (350 mL) was addedBu₄NBr (17.3 g, 53.7 mmol), Cs₂CO₃ (17.5 g, 53.7 mmol), and Pd(OAc)₂(2.01 g, 8.96 mmol). The flask was degassed with two cycles of vacuum/N₂backfill and heated to reflux overnight, under an atmosphere of N₂.After cooling to rt, the mixture was filtered through celite, elutingwith THF, and concentrated. The residue was dissolved in 500 mL EtOAc,washed with 400 mL sat. aq. NaHCO₃, 2×400 mL water, and 400 mL of brine.The organic fraction was dried with Na₂SO₄, filtered, and concentratedunder vacuum. The residue was purified by silica gel chromatography(CH₂Cl₂/triethylamine), giving 5 (15.1 g, 68%) as a foamy solid. ¹H NMR(400 MHz, DMSO-d₆) δ ppm 3.74 (s, 6H) 3.87 (s, 3H) 4.11 (s, 2H) 5.23 (s,2H) 6.89-6.96 (m, 4H) 7.01 (d, J=1.52 Hz, 1H) 7.08 (dd, J=8.08, 1.52 Hz,1H) 7.22-7.27 (m, 1H) 7.29-7.33 (m, 4H) 7.33-7.38 (m, 2H) 7.41-7.46 (m,2H) 7.88-7.93 (m, 2H) 7.93-7.99 (m, 2H)

Lithium aluminum hydride (43.4 mL of 1.0 M suspension in THF, 43.4 mmol)was added to a solution of compound 5 (12.0 g, 19.3 mmol) in THF (150mL) at 0° C. After 2 hours at 0° C., the reaction mixture was quenchedby dropwise addition of 20 mL EtOAc, with stirring at 0° C. for 10 min.1.65 mL H₂O, 1.65 mL 20% aq. NaOH, and 4.95 mL H₂O were addedsuccessively. The mixture was then stirred at rt for 1 h, dried withNa₂SO₄, filtered through celite, and concentrated under vacuum. Theresidue was purified by silica gel chromatography (ethylacetate/heptane/triethylamine), giving 6 (8.47 g, 81%) as a foamy solid.¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.75 (s, 6H) 4.07 (s, 2H) 4.52 (d,J=5.56 Hz, 2H) 5.13 (s, 2H) 5.21-5.25 (m, 1H) 6.89-6.95 (m, 4H) 6.96 (d,J=1.52 Hz, 1H) 7.03 (dd, J=8.08, 1.52 Hz, 1H) 7.22 (s, 1H) 7.23-7.28 (m,1H) 7.29-7.33 (m, 4H) 7.33-7.38 (m, 3H) 7.41-7.46 (m, 2H) 7.76 (d,J=7.58 Hz, 1H) 7.81 (d, J=8.08 Hz, 1H). MS (ESI+) m/z: calcd forC₃₆H₃₂O₅544.2. found 545.2 [MH⁺].

Dimethylaminopyridine (0.124 g, 1.02 mmol) was added to a solution ofcompound 6 (0.554 g, 1.02 mmol) in pyridine (5 mL) at rt under argon.Succinic anhydride 7 (0.204 g, 2.03 mmol) was added and the solution wasstirred at rt for 6 h. 0.5 mL H₂O was added, and the solution wasstirred for 30 min. 100 mL of CH₂Cl₂ was added, and the solution waswashed 1× with 50 mL cold 10% aq. citric acid and 2× each with 50 mL ofwater. The aqueous fractions were reextracted with 1×50 mL of CH₂Cl₂.The combined organic fractions were dried with Na₂SO₄, filtered,concentrated under vacuum and then diluted/concentrated 2× with toluene.The residue was purified by silica gel chromatography(dichloromethane/methanol/triethylamine) (49:1/1%), giving 8 (0.78 g,103%) as a foamy solid. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.45 (t, J=7.20Hz, 2.5H) 2.52-2.60 (m, 2H) 2.65-2.71 (m, 2H) 3.60 (q, J=7.33 Hz, 1.7H)3.79 (s, 6H) 4.16 (s, 2H) 5.12 (s, 2H) 5.13 (s, 2H) 6.82-6.87 (m, 4H)7.03 (dd, J=8.08, 1.26 Hz, 1H) 7.08 (s, 1H) 7.17 (s, 1H) 7.19-7.25 (m,1H) 7.28-7.33 (m, 2H) 7.33-7.37 (m, 1H) 7.38-7.44 (m, 4H) 7.49-7.54 (m,2H) 7.66 (t, J=7.83 Hz, 2H)

2.K. Synthesis of X066 Succinate Ester

To a 40 mL vial with septa were added 4-bromo-3-(bromomethyl)phenol (1,0.360 g, 1.25 mmol), methyl 3-(bromomethyl)benzoate (2, 0.856 g, 3.74mmol), K₂CO₃ (0.516 g, 3.74 mmol), and acetone (6 mL). The vial wasevacuated/N₂ backfilled 2×, and heated at 50° C. for 20 h. after coolingto rt, the mixture was filtered washing with CH₂Cl₂, and concentratedunder vacuum. The residue was purified by silica gel chromatography(ethyl acetate/heptane), giving 3 (0.391 g, 62%) as a white solid. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 3.88 (s, 3H) 4.67 (s, 2H) 5.21 (s, 2H) 6.98(dd, J=8.59, 3.03 Hz, 1H) 7.35 (d, J=3.03 Hz, 1H) 7.52-7.58 (m, 2H) 7.72(d, J=8.08 Hz, 1H) 7.91-7.95 (m, 1H) 8.05 (s, 1H)

Synthesis of compound 4 is described in the synthesis of X063. To a 40mL vial with a septa was added compounds 3 (0.390 g, 0.763 mmol), 4(0.390 g, 0.915 mmol), K₂CO₃ (0.316 g, 2.29 mmol) and acetone (4 mL).The vial was sealed and the contents were evacuated/N₂ backfilled 2×.The vial was then heated at 60° C. for 17 h. After cooling to rt, themixture was filtered washing with CH₂Cl₂, and concentrated under vacuum.The residue was purified by silica gel chromatography (ethylacetate/heptane/triethylamine), giving 5 (0.448 g, 77%) as a foamysolid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.74 (s, 6H) 3.85 (s, 3H) 4.10(s, 2H) 5.08 (s, 2H) 5.20 (s, 2H) 6.87-6.92 (m, 4H) 6.92-7.02 (m, 4H)7.19-7.26 (m, 3H) 7.26-7.35 (m, 7H) 7.39-7.45 (m, 2H) 7.50 (t, J=7.83Hz, 1H) 7.56 (d, J=8.59 Hz, 1H) 7.68 (d, J=7.58 Hz, 1H) 7.90 (d, J=7.58Hz, 2H) 8.02 (s, 1H)

To compound 5 (0.540 g, 0.569 mmol) in a vial with a septa, was addeddimethoxyethane (5.7 mL), Bu₄NBr (0.275 g, 0.853 mmol), Cs₂CO₃ (0.278 g,0.853 mmol), and Pd(OAc)₂ (0.026 g, 0.11 mmol). The vial was sealed,degassed with two cycles of vacuum/N₂ backfill, and heated at 90° C.overnight.-33% conversion was observed after 17 h by LCMS. An additional0.100 g of Pd(OAc)₂ (0.44 mmol) was added, and the reaction wascontinued for an additional 24 h. After cooling to rt, the mixture wasfiltered through celite eluting with EtOAc. The solution was then washed1× each with aq. sat. NaHCO₃, water and brine. The organic portion wasdried with Na₂SO₄, filtered, and concentrated under vacuum. The residuewas purified by silica gel chromatography (ethylacetate/heptane/triethylamine), giving 6 (0.105 g, 27%) as a foamysolid. ¹H NMR (400 MHz, DMSO-d6) δ ppm 3.75 (s, 6H) 3.87 (s, 3H) 4.08(s, 2H) 5.09 (s, 2H) 5.24 (s, 2H) 6.88-6.95 (m, 5H) 6.95-7.00 (m, 2H)7.05 (dd, J=8.34, 2.78 Hz, 2H) 7.21-7.26 (m, 2H) 7.29-7.36 (m, 6H)7.39-7.46 (m, 2H) 7.55 (t, J=7.83 Hz, 1H) 7.69-7.77 (m, 3H) 7.92 (d,J=8.08 Hz, 1H) 8.05 (s, 1H)

Compound 6 (0.135 g, 0.199 mmol) in THF (2 mL) was cooled to 0° C.,under an atmosphere of N₂. A 1M suspension of LAH in THF (0.477 mL,0.477 mmol) was added dropwise, and the solution was stirred at 0° C.for 3 h. 1 mL EtOAc was added dropwise, and the solution was stirred at0° C. for 20 min. 0.018 mL H₂O, 0.018 mL 20% aq. NaOH, and 0.054 mL H₂Owere added successively, and the mixture was stirred at rt for 1 h. themixture was dried with Na₂SO₄, filtered through celite washing withEtOAc, and concentrated under vacuum. The residue was purified by silicagel chromatography (ethyl acetate/heptane/triethylamine), giving 7(0.110 g, 85%) as a foamy solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.75(s, 6H) 4.08 (s, 2H) 4.52 (d, J=5.56 Hz, 2H) 5.04 (t, J=5.81 Hz, 1H)5.08 (s, 2H) 5.14 (s, 2H) 6.89-6.94 (m, 5H) 6.95 (d, J=2.53 Hz, 1H) 6.98(dd, J=8.08, 1.52 Hz, 1H) 7.03 (dd, J=8.59, 2.53 Hz, 1H) 7.24 (t, J=7.33Hz, 1H) 7.26-7.37 (m, 9H) 7.40-7.46 (m, 3H) 7.72 (d, J=8.08 Hz, 2H). MS(ESI+) m/z: calcd for C₄₃H₃₈O₆650.3. found 303.4 [DMT⁺].

Dimethylaminopyridine (0.019 g, 0.157 mmol) was added to a solution ofcompound 7 (0.102 g, 0.157 mmol) in pyridine (3 mL) at rt under argon.Succinic anhydride 8 (0.031 g, 0.313 mmol) was added, and the solutionwas stirred at rt for 16 h. 0.5 mL of H₂O was added and the solution wasstirred for 30 min. 50 mL of CH₂Cl₂ was added, the solution was washed1× with 25 mL cold 10% aq. citric acid and 2× each with 25 mL of water.The aqueous fractions were reextracted with 1×25 mL of CH₂Cl₂. Theorganic fractions were dried with Na₂SO₄, filtered, concentrated undervacuum, and diluted/concentrated 2× with toluene. The residue waspurified by silica gel chromatography(dichloromethane/methanol/triethylamine) (49:1/1%), giving 9 (0.10 g,76%) as an oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.46 (t, J=7.33 Hz, 2.1H)2.57 (t, J=6.82 Hz, 2H) 2.68 (t, J=6.82 Hz, 2H) 3.61 (q, J=7.16 Hz,1.4H) 3.80 (s, 6H) 4.15 (s, 2H) 5.09 (s, 2H) 5.09 (s, 2H) 5.15 (s, 2H)6.78 (d, J=2.53 Hz, 1H) 6.82-6.88 (m, 4H) 6.95-7.04 (m, 2H) 7.06 (s, 1H)7.18-7.25 (m, 1H) 7.28-7.36 (m, 3H) 7.36-7.46 (m, 7H) 7.50-7.55 (m, 2H)7.61 (dd, J=8.34, 2.27 Hz, 2H)

2.L. Synthesis of X051 Succinate

The synthesis of compound 1 is described in the synthesis of X063.Compound 1 (6.70 g, 12.3 mmol) was dissolved in THF (123 mL),evacuated/N₂ purged 2×, and cooled to 0° C. A 60% dispersion of NaH inmineral oil was added (0.886 g, 36.9 mmol), and the mixture was stirredat 0° C. for 20 min. Methyl 3-(bromomethyl)benzoate (2, 3.38 g, 14.8mmol) was then added, and the mixture was stirred at rt for 20 h. Thereaction mixture was then diluted with 400 mL of EtOAc and washed 1×with 400 mL sat. aq. NaHCO₃. The aqueous layer was back-extracted with200 mL of EtOAc, and the combined organic layers were washed 1× eachwith 400 mL water and brine. The organic portion was dried with Na₂SO₄,filtered, and concentrated under vacuum. The residue was purified bysilica gel chromatography (ethyl acetate/heptane/triethylamine), giving3 (6.55 g, 77%) as a foamy solid. The product contained ˜13% of thecorresponding ethyl ester that was carried forward to the next step asan equivalent precursor. Methyl ester: ¹H NMR (400 MHz, DMSO-d₆) δ ppm1.32 (t, J=7.07 Hz, 0.38H) 3.74 (s, 6H) 3.86 (s, 2.52H) 4.08 (s, 2H)4.32 (q, J=7.07 Hz, 0.25H) 4.58 (s, 2H) 4.64 (s, 2H) 5.14 (s, 2H) 6.93(d, J=8.59 Hz, 4H) 6.97 (d, J=1.52 Hz, 1H) 7.04 (dd, J=8.08, 1.52 Hz,1H) 7.25 (t, J=7.33 Hz, 1H) 7.28 (s, 1H) 7.31 (d, J=9.09 Hz, 4H)7.33-7.41 (m, 3H) 7.44 (d, J=7.58 Hz, 2H) 7.53 (t, J=7.83 Hz, 1H) 7.66(d, J=8.08 Hz, 1H) 7.80 (d, J=8.08 Hz, 1H) 7.83 (d, J=8.08 Hz, 1H) 7.90(d, J=7.58 Hz, 1H) 7.97 (s, 1H)

Compound 3 in THF was cooled to 0° C. and placed under an atmosphere ofN₂. A 1M suspension of LAH in THF (22.5 mL, 22.5 mmol) was addeddropwise, and the solution was stirred at 0° C. for 2 h. 1 mL EtOAc wasadded dropwise, and the solution was stirred at 0° C. for 20 min. Then0.86 mL H₂O, 0.86 mL 20% aq. NaOH, and 2.58 mL H₂O were addedsuccessively. The mixture was stirred at rt for 1 h, dried with Na₂SO₄,filtered through celite washing with EtOAc, and concentrated undervacuum. The residue was purified by silica gel chromatography (ethylacetate/heptane/triethylamine), giving 4 (5.98 g, 96%) as a foamy solid.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.74 (s, 6H) 4.08 (s, 2H) 4.50 (d,J=6.06 Hz, 2H) 4.55 (s, 2H) 4.55 (s, 2H) 5.14 (s, 2H) 5.18 (t, J=5.81Hz, 1H) 6.93 (d, J=8.59 Hz, 4H) 6.97 (d, J=1.01 Hz, 1H) 7.01-7.06 (m,1H) 7.21-7.28 (m, 4H) 7.28-7.33 (m, 5H) 7.33-7.40 (m, 4H) 7.41-7.46 (m,2H) 7.80 (d, J=8.08 Hz, 1H) 7.83 (d, J=8.08 Hz, 1H). MS (ESI+) m/z:calcd for C₄₄H₄₀O₆664.3. found 665.3 [MH⁺].

Dimethylaminopyridine (0.37 g, 3.01 mmol) was added to a solution ofcompound 4 (2.00 g, 3.01 mmol) in pyridine (15 mL) at rt under argon.Succinic anhydride 7 (0.60 g, 6.02 mmol) was added, and the solution wasstirred at rt for 17 h. 1 mL of H₂O was added and the solution wasstirred for 1 h. 100 mL of CH₂Cl₂ was added, and the solution was washed1× with 50 mL cold 10% aq. citric acid and 2× each with 50 mL of water.The aqueous fractions were reextracted with 1×50 mL of CH₂Cl₂. Thecombined organic fractions were dried with Na₂SO₄, filtered,concentrated under vacuum, and diluted/concentrated 2× with toluene. Theresidue was purified by silica gel chromatography(dichloromethane/methanol/triethylamine) (39:1/1%), giving 6 (2.48 g,95%) as an oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.17 (t, J=7.33 Hz, 14.2H)2.56 (t, J=6.69 Hz, 2H) 2.68 (t, J=7.45 Hz, 2H) 2.84 (q, J=7.33 Hz,9.5H) 3.80 (s, 6H) 4.16 (s, 2H) 4.57 (s, 2H) 4.58 (s, 2H) 5.14 (s, 2H)5.14 (s, 2H) 6.82-6.88 (m, 4H) 7.01-7.05 (m, 1H) 7.09 (s, 1H) 7.18 (s,1H) 7.19-7.25 (m, 1H) 7.28-7.34 (m, 5H) 7.34-7.38 (m, 2H) 7.39-7.44 (m,4H) 7.49-7.55 (m, 2H) 7.68 (dd, J=7.96, 4.42 Hz, 2H)

2.M. Synthesis of X097 Succinate Ester

(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-bromophenyl)methanol(2)

A solution of 5-bromo-1,3-dihydroxymethyl benzene 1 (3.00 g, 13.8 mmol),4,4′-dimethoxytrityl chloride (4.68 g, 13.8 mmol) in pyridine (60 mL)was stirred at room temperature for 16 h. The reaction mixture waspartitioned between EtOAc and water. The EtOAc layer was dried overanhydrous Na₂SO₄ and evaporated. The crude product was purified by flashchromatography eluting with 1% Et₃N in 5-30% EtOAc/Heptane to provide2.57 g (36%) of 2. MS (ESI+) m/z: calcd for C₂₉H₂₇BrO₄ 518.1. found303.5 [DMT]⁺. ¹H NMR (400 MHz, CDCl₃) b 7.54-7.48 (m, 2H), 7.47-7.37 (m,6H), 7.36-7.29 (m, 2H), 7.27-7.21 (m, 2H), 6.87 (d, J=8.8 Hz, 4H), 4.67(d, J=6.0 Hz, 2H), 4.22 (s, 2H), 3.82 (s, 6H), 1.67 (t, J=6.0 Hz, 1H).

(3′-(benzyloxy)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-[1,1′-biphenyl]-3-yl)methanol(4)

To a mixture of bromide 2 (0.50 g, 0.963 mmol), 3-benzyloxybenzeneboronic acid 3 (0.263 g, 1.155 mmol) and Pd(PPh₃)₄ (0.111 g, 0.096 mmol)in 1,4-dioxane (6 mL) under nitrogen atmosphere was added 2M aq. Na₂CO₃(1.44 mL). The mixture was heated at reflux overnight. The reaction isthen cooled to room temperature and partitioned between EtOAc and sat.aq. NaHCO₃. The organic layer was evaporated and the crude product waspurified by flash chromatography eluting with 1% Et₃N in 5-30%EtOAc/Heptane to provide 0.454 g (70%) of 4. MS (ESI+) m/z: calcd forC₄₂H₃₈O₅622.3. found 303.5 [DMT]⁺. ¹H NMR (400 MHz, DMSO) δ 7.52-7.43(m, 5H), 7.41-7.29 (m, 12H), 7.27-7.16 (m, 3H), 7.01 (m, 1H), 6.95-6.86(m, 4H), 5.18 (s, 2H), 5.07 (t, J=5.8 Hz, 1H), 4.57 (d, J=5.8 Hz, 2H),4.18 (s, 2H), 3.74 (s, 6H).

To a solution of 452 mg (0.726 mmol) 4 and 89 mg (0.726 mmol)N,N-dimethylaminopyridine (DMAP) in 5 mL dry pyridine under argon wasadded 145 mg (1.45 mmol) succinic anhydride (5). The reaction mixturewas stirred at room temperature for 18 h and then 0.5 mL water wasadded. Stirring was continued for 30 min. The reaction mixture wasdiluted with 100 mL dichloromethane and washed with 50 mL ice-cold 10%aqueous citric acid and water (2×50 mL). The aqueous layers werereextracted with 50 mL dichloromethane. The combined organic layers weredried over Na₂SO₄ and evaporated. The remaining oil was coevaporatedtwice with toluene and the crude product purified by silica gelchromatography (dichloromethane/methanol/triethylamine 94:5:1) to give510 mg (0.619 mmol, 85%) 6 as a colorlessfoam. ¹H NMR (400 MHz, CDCl₃):1.22 (t, J=7.3 Hz, 9H), 2.57-2.59 (m, 2H), 2.67-2.69 (m, 2H), 2.97 (q,J=7.3 Hz, 6H), 3.79 (s, 6H), 4.24 (s, 2H), 5.14 (s, 2H), 5.18 (s, 2H),5.72 (s br., 1H), 6.84-6.88 (m, 4H), 6.98 (ddd, J=0.7, 2.2, 8.2 Hz, 1H),7.19-7.54 (m, 20H).

2.N. Synthesis of X098 Succinate Ester

(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-bromophenyl)methanol(2)

Same as Above

A solution of 5-bromo-1,3-dihydroxymethyl benzene 1 (3.00 g, 13.8 mmol),4,4′-dimethoxytrityl chloride (4.68 g, 13.8 mmol) in pyridine (60 mL)was stirred at room temperature for 16 h. The reaction mixture waspartitioned between EtOAc and water. The EtOAc layer was dried overanhydrous Na₂SO₄ and evaporated. The crude product was purified by flashchromatography eluting with 1% Et₃N in 5-30% EtOAc/Heptane to provide2.57 g (36%) of 2. MS (ESI+) m/z: calcd for C₂₉H₂₇BrO₄ 518.1. found303.5 [DMT]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.54-7.48 (m, 2H), 7.47-7.37 (m,6H), 7.36-7.29 (m, 2H), 7.27-7.21 (m, 2H), 6.87 (d, J=8.8 Hz, 4H), 4.67(d, J=6.0 Hz, 2H), 4.22 (s, 2H), 3.82 (s, 6H), 1.67 (t, J=6.0 Hz, 1H).

(5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-[1,1′-biphenyl]-3-yl)methanol(4)

To a mixture of the bromide 2 (0.500 g, 0.960 mmol), benzene boronicacid 3 (0.141 g, 1.16 mmol), and Pd(PPh₃)₄ (0.111 g, 0.096 mmol) in1,4-dioxane (6 mL) under nitrogen atmosphere was added 2M aq. Na₂CO₃(1.44 mL). The mixture was heated at reflux overnight. The reaction wasthen cooled to room temperature and partitioned between EtOAc and sat.aq. NaHCO₃. The organic layer was evaporated and the crude product waspurified by flash chromatography eluting with 1% Et₃N in 5-30%EtOAc/Heptane to provide 0.380 g (76%) of 4. MS (ESI+) m/z: calcd forC₃₅H₃₂O₄516.2. found 303.5 [DMT]⁺. ¹H NMR (400 MHz, DMSO) δ 7.61 (dd,J=8.3, 1.3 Hz, 2H), 7.51-7.42 (m, 5H), 7.39-7.28 (m, 9H), 7.27-7.20 (m,1H), 6.95-6.86 (m, 4H), 5.08 (t, J=5.8 Hz, 1H), 4.57 (d, J=5.8 Hz, 2H),4.19 (s, 2H), 3.74 (s, 6H).

To a solution of 380 mg (0.736 mmol) 4 and 90 mg (0.736 mmol)N,N-dimethylaminopyridine (DMAP) in 5 mL dry pyridine under argon wasadded 147 mg (1.47 mmol) succinic anhydride (5). The reaction mixturewas stirred at room temperature for 18 h and then 0.5 mL water wasadded. Stirring was continued for 30 min. The reaction mixture wasdiluted with 100 mL dichloromethane and washed with 50 mL ice-cold 10%aqueous citric acid and water (2×50 mL). The aqueous layers werereextracted with 50 mL dichloromethane. The combined organic layers weredried over Na₂SO₄ and evaporated. The remaining oil was coevaporatedtwice with toluene and the crude product purified by silica gelchromatography (dichloromethane/methanol/triethylamine 94:5:1) to give460 mg (0.641 mmol, 87%) 6 as an off-white foam. ¹H NMR (400 MHz,CDCl₃): 1.22 (t, J=7.2 Hz, 9H), 2.59 (t, J=7.1 Hz, 2H), 2.71 (t, J=7.1Hz, 2H), 2.94 (q, J=7.2 Hz, 6H), 3.81 (s, 6H), 4.26 (s, 2H), 5.20 (s,2H), 6.04 (s br., 1H), 6.85-6.89 (m, 4H), 7.22-7.55 (m, 15H), 7.62 (d,J=7.9 Hz, 2H).

2.O. Synthesis of siRNA Conjugated with X109ss-siRNA=antisense single strand sequence used inconjugation=U002pUpApApU004pU004pApU004pCpU004pApU004pU004pCpCpGpU005pA005pC027Where C0027 is ribitol

A mixture of 1 (100 mg, 0.361 mmol), N-hydroxysuccinimide (83 mg, 0.721mmol) and DCC (149 mg, 0.721 mmol) in DCE (4 mL) was stirred at RT for12 h. The reaction mixture was quenched with sat. aq. NaHCO₃ (4 mL). Theorganic layer was separated from the water layer, and was washed withwater (1 mL) and brine (1 mL). The organic solvent was removed undervacuum. The crude product was purified by recrystallization frommethanol to give 2 (27.7 mg, 0.074 mmol) in 21% yield. ESI MS (m/z,MH⁺): 375.4; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.85 (d, J=5.52 Hz,4H) 2.89 (s, 3H) 3.94 (s, 2H) 7.31-7.48 (m, 4H) 7.49-7.64 (m, 3H) 7.71(t, J=6.78 Hz, 1H) 8.12 (br. s., 1H).

To 2 (2.23 mg, 5.96 umol) in DMSO (73.2 uL) was added a freshly preparedss-siRNA-(CH₂)₃—NH₂ solution (3.66 mg, 0.596 umol in 73.2 uL PBS 8.5buffer). The reaction mixture was vortexed and sat at RT for 30 min. Thecrude product was purified by HPLC with 5-60% 100 mM triethylammoniumacetate in acetonitrile/water to afford 3 (1.09 mg, 0.164 umol) in 27.5%yield. TOF MS (ES⁻): 6403.

2.P. Synthesis of siRNA Conjugated with X110

A mixture of 1 (500 mg, 1.40 mmol), Pd (30% on carbon, 24.9 mg, 0.070mmol), and acetic acid (80 ul, 1.40 mmol) in methanol (15 mL) wasstirred at RT under H₂ (1 atm) for 12 h. The reaction mixture wasfiltered to remove Pd/C. To the solution was added aq. 1M NaOH (3 mL),and the resulting mixture was heated at 60° C. for 12 h. The mixture wascooled to RT and neutralized with aq. 1M HCl to give form a precipitate.The precipitate was collected by vacuum filtration and dried in the ovento give 2 (166 mg, 0.63 mmol) with 45% yield. ESI MS (m/z, MH⁺): 264.4.¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.58 (s, 2H) 7.18-7.39 (m, 3H) 7.44-7.65(m, 4H) 7.75 (ddd, J=8.28, 6.78, 1.51 Hz, 1H) 8.01-8.20 (m, 1H) 8.91 (s,1H) 12.47 (s, 1H).

A mixture of 2 (87.6 mg, 0.333 mmol), N-hydroxysuccinimide (77.0 mg,0.665 mmol) and DCC (137 mg, 0.665 mmol) in DCM (4 mL) was stirred at RTfor 12 h. The reaction mixture was quenched with sat. aq. NaHCO₃ (4 mL).The organic layer was separated from the water layer, and was washedwith water (1 mL) and brine (1 mL). The organic solvent was removedunder vacuum. The crude product was purified by recrystallization frommethanol to give 3 (27.7 mg, 0.074 mmol) in 49% yield. ESI MS (m/z,MH⁺): 361.2. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.79 (br. s., 4H) 4.05 (s,2H) 7.29-7.37 (m, 2H) 7.40 (s, 1H) 7.50-7.64 (m, 4H) 7.80 (s, 1H) 8.10(s, 1H) 9.02 (s, 1H).

To 3 (1.76 mg, 4.88 umol) in DMSO (240 uL) was added a freshlyss-siRNA-(CH₂)₃—NH₂ solution (2 mg, 0.325 umol in 40 uL PBS 8.5 buffer).The reaction mixture was vortexed and sat at RT for 30 min. The crudeproduct was purified by HPLC with 5-60% 100 mM triethylammonium acetatein acetonitrile/water to afford 4 (0.526 mg, 0.082 umol) in 25% yield.TOF MS (ES⁻): 6388.

2. Q. Synthesis of siRNA Conjugated with X1111 is commercial, but synthesis is not known in the literature

A mixture of 1 (81 mg, 0.308 mmol), N-hydroxysuccinimide (70.8 mg, 0.615mmol) and DCC (127 mg, 0.615 mmol) in DCM (4 mL) was stirred at RT for12 h. The reaction mixture was quenched with sat. aq. NaHCO₃ (4 mL). Theorganic layer was separated from the water layer, and was washed withwater (1 mL) and brine (1 mL). The organic solvent was removed undervacuum. The crude product was purified by recrystallization frommethanol to give 2 (43.7 mg, 0.121 mmol) in 39% yield. ESI MS (m/z,MH⁺): 361.4. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 2.82 (s, 4H) 4.07 (s,2H) 7.36-7.42 (m, 2H) 7.46-7.51 (m, 1H) 7.55-7.64 (m, 3H) 7.70-7.77 (m,2H) 8.20 (dt, J=4.52, 2.26 Hz, 1H) 9.29 (s, 1H).

To 2 (2.35 mg, 6.51 umol) in DMSO (240 uL) was added a freshlyss-siRNA-(CH₂)₃—NH₂ solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer).The reaction mixture was vortexed and sat at RT for 30 min. The crudeproduct was purified by HPLC with 5-60% 100 mM triethylammonium acetatein acetonitrile/water to afford 3 (0.68 mg, 0.082 umol) in 33% yield.TOF MS (ES⁻): 6390.

2.R. Synthesis of siRNA Conjugated with X112

A mixture of 1 (100 mg, 0.325 mmol), tert-butylchlorodimethylsilane (108mg, 0.716 mmol) and imidazole (91 mg, 1.33 mmol) in DMF (4 mL) wasstirred at RT for 48 h. The reaction mixture was quenched with water (4mL) and extracted with ethyl acetate (3×5 mL). The combined organiclayers were washed with water and brine, and dried over sodium sulfate.The organic solvent was then removed under vacuum. The crude product waspurified by silica chromatography with 0-50% ethyl acetate/heptane togive 2 (55 mg, 0.13 mmol) in 40% yield. ESI MS (m/z, MH⁺): 422.2. ¹H NMR(400 MHz, CHLOROFORM-d) δ ppm 0.04 (m, 6H) 0.88-0.93 (m, 9H) 3.59 (t,J=6.78 Hz, 2H) 3.71 (s, 2H) 4.09 (t, J=6.78 Hz, 2H) 7.28-7.45 (m, 4H)7.51-7.61 (m, 3H) 7.63-7.68 (m, 1H) 9.00 (s, 1H).

A mixture of N-hydroxysuccinimide (2.73 mg, 0.024 mmol), 2 (5.0 mg,0.012 mmol), and DIC (2 mg, 0.325 umol) in DMSO (200 uL) was stirred atRT for 12 h. 10 uL reaction mixture was diluted with 190 uL DMSO. To theresulting solution was added a freshly ss-siRNA-(CH₂)₃—NH₂ solution(2.19 mg, 0.356 umol in 80 ul PBS 8.5 buffer). The reaction mixture wasvortexed and sat at RT for 2 h. The crude product was purified by HPLCwith 10-40% 100 mM triethylammonium acetate in acetonitrile/water toafford 3 (0.79 mg, 0.123 umol) in 35% yield. TOF MS (ES⁻): 6435.

2.S. Synthesis of siRNA Conjugated with X113

1 is commercial and synthesis is known in the literature. Zhang, Yan etal. From PCT Int. Appl., 2010083384, 22 Jul. 2010.

A mixture of N-hydroxysuccinimide (6.18 mg, 0.054 mmol), 1 (5.0 mg,0.027 mmol), and DIC (6.77 mg, 0.054 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₃—NH₂solution (2.46 mg, 0.401 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2 (0.24 mg, 0.038 umol) in 10% yield. TOFMS (ES⁻): 6315.

2. S. 1. General Procedure for the High Density Loading of ControlledPore Glass Supports with PAZ Ligand Succinates

In an Erlenmeyer flask 1.00 mmol PAZ ligand succinate salt 1 wasdissolved in 50 mL dry acetonitrile under argon. To this solution 353 mg(1.10 mmol)O-(1H-benzo-1,2,3-triazol-1-yl)-N,N,N′,N′-tetramethyl-uroniumtetrafluoroborate (TBTU) was added and the solution shaken for 10 min.Then 10 g long chain alkylamine controlled pore glass (LCAA/CNA-600-CPG,PrimeSynthesis, 2) was added and the reaction mixture gently agitatedfor 5 min. Finally, 0.685 mL (517 mg, 4.00 mmol) Hünig's base was addedand the flask gently shaken for 24 h on an orbital shaker. Loadingdensity was assessed by detritylating an aliquote of the CPG (3-5 mg CPGwashed with acetonitrile, dried in vacuo, added to 25 mL 3%dichloroacetic acid in dichloromethane (v/v), absorbance at 504 nmdetermined). If loading density was in the desired range (60-90micromol/g), the CPG was filtered off and washed extensively withacetonitrile. Underivatized amino groups were capped by treating the CPGwith x mL each of a mixture of acetic anhydride/2,6-lutidine/THF 1:1:8(v/v/v) and a solution of 1-methylimidazole in THF 16:84 (v/v). Themixture was gently shaken for 15 min at room temperature. Then the CPGwas filtered off, washed with acetonitrile and dried under vacuumovernight. Loading density was determined again as above. Loading yieldsfor the succinates in examples 1-6 were in the range of 64-75micromol/g.

2. T. Synthesis of siRNA Conjugated with X1011

A mixture of N-hydroxysuccinimide (2.489 mg, 0.022 mmol), 1 (3.0 mg,0.011 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2.00 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6418.

2. U. Synthesis of siRNA Conjugated with X1012 and X1018

A mixture of 1 (500 mg, 1.40 mmol), Pd (30% on carbon, 24.9 mg, 0.070mmol), and acetic acid (80 ul, 1.40 mmol) in methanol (15 mL) wasstirred at RT under H₂ (1 atm) for 12 h. The reaction mixture wasfiltered to remove Pd/C. To the solution was added aq. 1M NaOH (3 mL),and the resulting mixture was heated at 60° C. for 12 h. The mixture wascooled to RT and neutralized with aq. 1M HCl to give form a precipitate.The precipitate was collected by vacuum filtration and dried in the ovento give 2 (166 mg, 0.63 mmol) with 45% yield. ESI MS (m/z, MH⁺): 264.4.¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.58 (s, 2H) 7.18-7.39 (m, 3H) 7.44-7.65(m, 4H) 7.75 (ddd, J=8.28, 6.78, 1.51 Hz, 1H) 8.01-8.20 (m, 1H) 8.91 (s,1H) 12.47 (s, 1H).

A mixture of N-hydroxysuccinimide (2.489 mg, 0.022 mmol), 2 (2.85 mg,0.011 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2.00 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 3. ESI MS (ES⁺): 6405.

2. U. Synthesis of siRNA Conjugated with X1018

A mixture of N-hydroxysuccinimide (2.483 mg, 0.022 mmol), 2 (2.84 mg,0.011 mmol), and DIC (2.72 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₅—NH₂solution (2.00 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 4.

2. V. Synthesis of siRNA Conjugated with X1013

A mixture of N-hydroxysuccinimide (2.489 mg, 0.022 mmol), 2 (2.85 mg,0.011 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2.00 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6404.

2.W. Synthesis of siRNA Conjugated with X1019

A mixture of 1 (100 mg, 0.325 mmol), tert-butylchlorodimethylsilane (108mg, 0.716 mmol) and imidazole (91 mg, 1.33 mmol) in DMF (4 mL) wasstirred at RT for 48 h. The reaction mixture was quenched with water (4mL) and extracted with ethyl acetate (3×5 mL). The combined organiclayers were washed with water and brine, and dried over sodium sulfate.The organic solvent was then removed under vacuum. The crude product waspurified by silica chromatography with 0-50% ethyl acetate/heptane togive 2 (55 mg, 0.13 mmol) in 40% yield. ESI MS (m/z, MH⁺): 422.2. ¹H NMR(400 MHz, CHLOROFORM-d) δ ppm 0.04 (m, 6H) 0.88-0.93 (m, 9H) 3.59 (t,J=6.78 Hz, 2H) 3.71 (s, 2H) 4.09 (t, J=6.78 Hz, 2H) 7.28-7.45 (m, 4H)7.51-7.61 (m, 3H) 7.63-7.68 (m, 1H) 9.00 (s, 1H).

A mixture of N-hydroxysuccinimide (2.48 mg, 0.022 mmol), 2 (4.55 mg,0.011 mmol), and DIC (2.72 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL reaction mixture was diluted with 190 uL DMSO. Tothe resulting solution was added a freshly ss-siRNA-(CH₂)₃—NH₂ solution(2 mg, 0.324 umol in 80 ul PBS 8.5 buffer). The reaction mixture wasvortexed and sat at RT for 2 h. The crude product was purified by HPLCwith 10-40% 100 mM triethylammonium acetate in acetonitrile/water toafford 3. TOF MS (ES⁻): 6462.

2. X. Synthesis of siRNA Conjugated with X1015

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 1 (2.02 mg,0.011 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6327.

2.Y. Synthesis of siRNA Conjugated with X1020

A mixture of N-hydroxysuccinimide (2.48 mg, 0.022 mmol), 1 (2.02 mg,0.011 mmol), and DIC (2.72 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₅—NH₂solution (2 mg, 0.324 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6341.

2. Z. Synthesis of siRNA Conjugated with X1009

To AlCl₃ (1.19 g, 8.93 mmol, 40 ml Toluene solution) under N₂ was added4-methoxyaniline (1 g, 8.12 mmol, 10 ml Toluene solution) dropwise. BCl₃(8.12 ml, 8.12 mmol, 1 M solution in CH₂Cl₂) and Benzonitrile (2.51 g,24.36 mmol) were added to the above mixture subsequently. The resultingmixture was stirred at RT for 1 h, then heated at 110° C. for 6 hrs. Thereaction mixture was cooled to RT, to which aq. HCl (1 M, 13 ml) wasadded. The solution was then heated at 80° C. for 1 h. The solution wascooled to RT, and the organic layer and water layer were separated. Thewater layer was extracted with ethyl acetate (3×50 mL). The combinedorganic layers were washed with water and brine, and dried over sodiumsulfate. The organic solvent was then removed under vacuum. The crudeproduct was purified by silica chromatography with 0-40% ethylacetate/heptane to give 1 (273 mg, 1.2 mmol) in 15% yield. ESI MS (m/z,MH⁺): 227.3. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 3.66 (s, 3H) 6.80 (d,J=8.84 Hz, 1H) 6.93-7.05 (m, 2H) 7.40-7.49 (m, 2H) 7.49-7.58 (m, 1H)7.63-7.72 (m, 2H).

A mixture of 1 (269 mg, 1.18 mmol) and ethyl 4-chloro-4-oxobutanoate(214 mg, 1.3 mmol) in DCM (10 ml) was heated at 60° C. for 1 h. Thereaction mixture was cooled and quenched with aq. 1 M NaOH (5 ml).Organic layer and water layer were separated. The water layer wasextracted with dichloromethane (3×5 ml). The combined organic layerswere washed with water and brine, and dried over sodium sulfate. Theorganic solvent was then removed under vacuum. The crude product waspurified by silica chromatography with 0-60% ethyl acetate/heptane togive 2 (305 mg, 0.86 mmol) in 73% yield. ESI MS (m/z, MH⁺): 355.5. ¹HNMR (400 MHz, CHLOROFORM-d) δ ppm 1.26 (t, J=7.28 Hz, 3H) 2.74 (s, 4H)3.77 (s, 3H) 4.16 (q, J=7.03 Hz, 2H) 7.06 (d, J=3.01 Hz, 1H) 7.14 (dd,J=9.03, 3.01 Hz, 1H) 7.48-7.55 (m, 2H) 7.60-7.66 (m, 1H) 7.73-7.79 (m,2H) 8.50 (d, J=9.03 Hz, 1H) 10.45 (br. s., 1H).

A mixture of 2 (305 mg, 0.86 mmol) and sodium hydride (343 mg, 8.58mmol) in ethanol (10 ml) was heated at 80° C. for 2 h. The reactionmixture was cooled to RT and quenched with water (5 ml) then neutralizedwith aq. 1 M HCl (2 ml). The resulting solution was extracted with ethylacetate (3×10 mL). The combined organic layers were washed with waterand brine, and dried over sodium sulfate. The organic solvent was thenremoved under vacuum to give 3 (250 mg, 0.81 mmol) in 94% yield. ESI MS(m/z, MH⁺): 309.3. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 3.38 (s, 2H) 3.63(s, 3H) 6.51 (d, J=2.51 Hz, 1H) 7.20 (dd, J=9.03, 3.01 Hz, 1H) 7.28-7.47(m, 3H) 7.52-7.63 (m, 3H).

A solution of 3 (250 mg, 0.81 mmol) in POCl₃ (10 ml) was stirred at RTfor 2 h. POCl₃ was removed under vacuum, the resulting residue wasquenched with ethanol (20 ml). The solution was stirred at RT for 1 h,then ethanol was removed under vacuum. To the residue was addeddichloromethane (30 ml) and aq. 1 M NaOH (20 ml). Organic layer andwater layer were separated. The water layer was extracted withdichloromethane (2×20 ml). The combined organic layers were washed withwater, brine and dried over sodium sulfate. The organic solvent wasremoved under vacuum to give 4 (270 mg, 0.8 mmol) in 99% yield. ESI MS(m/z, MH⁺): 338.2. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.23-1.27 (m,3H) 3.48 (s, 2H) 3.66 (s, 3H) 4.04-4.22 (m, 2H) 6.55 (d, J=2.51 Hz, 1H)7.09-7.15 (m, 1H) 7.24 (d, J=9.03 Hz, 1H) 7.29-7.34 (m, 2H) 7.44-7.64(m, 3H) 10.49 (br. s., 1H).

A solution of 4 (270 mg, 0.8 mmol) in POCl₃ (10 ml) was heated at 110°C. for 12 h. POCl₃ was removed under vacuum. To the residue was addeddichloromethane (20 ml) and aq. 1 M NaOH (20 ml). The organic layer andwater layer were separated. The water layer was extracted withdichloromethane (2×20 ml). The combined organic layers were washed withwater, brine and dried over sodium sulfate. The organic solvent wasremoved under vacuum to give 5 (265 mg, 0.75 mmol) in 92% yield. ESI MS(m/z, MH⁺): 356.1. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.22-1.27 (m,3H) 3.70 (s, 5H) 4.17 (q, J=7.03 Hz, 2H) 6.62 (d, J=3.01 Hz, 1H)7.20-7.34 (m, 2H) 7.38 (dd, J=9.03, 2.51 Hz, 1H) 7.46-7.63 (m, 3H) 8.01(d, J=9.03 Hz, 1H).

A mixture of 5 (265 mg, 0.745 mmol), triethylamine (1.28 g, 12.66 mmol),and Pd/C (10%, 79 mg, 0.745 mmol) in ethanol (20 ml) was stirred underH₂ (1 atm) at RT for 12 h. The reaction mixture was filtered to removePd/C. The organic solvent was removed under vacuum to give 6 (182 mg,0.57 mmol) in 76% yield. ESI MS (m/z, MH⁺): 322.1. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.12 (t, J=7.15 Hz, 3H) 3.51 (s, 2H) 3.62 (s, 3H)4.01 (q, J=7.19 Hz, 2H) 6.61 (d, J=2.76 Hz, 1H) 7.18-7.31 (m, 3H)7.39-7.50 (m, 3H) 7.97 (d, J=9.29 Hz, 1H) 8.68 (s, 1H).

A mixture of 6 (50 mg, 0.16 mmol), aq. 1 M LiOH (0.17 ml, 0.17 mmol) inDioxane (1 ml) was stirred at RT for 48 hrs. A precipitation from thereaction mixture was filtered and dried to give 7 (32 mg, 0.107 mmol) in69% yield as lithium salt. ESI MS (m/z, MH⁺): 294.2. ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 3.48 (s, 2H) 3.63-3.73 (m, 3H) 6.75 (d, J=2.51 Hz,1H) 7.28-7.43 (m, 3H) 7.48-7.64 (m, 3H) 7.94 (d, J=9.03 Hz, 1H) 8.73 (s,1H).

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 7 (3.18 mg,0.011 mmol), and DIC (2.74 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₃—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 8. TOF MS (ES⁻): 6422.

2. AA. Synthesis of siRNA Conjugated with X1016

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 7 (3.18 mg,0.011 mmol), and DIC (2.74 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 9. TOF MS (ES⁻): 6434.

2. BB. Synthesis of siRNA Conjugated with X1021

A mixture of N-hydroxysuccinimide (2.48 mg, 0.022 mmol), 7 (3.16 mg,0.011 mmol), and DIC (2.72 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₅—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 10. TOF MS (ES⁻): 6448.

2. CC. Synthesis of siRNA Conjugated with X1010

To BCl₃ (10.74 ml, 10.74 mmol, 1 M solution in CH₂Cl₂) under N₂ wasadded aniline (1 g, 10.74 mmol, 10 ml Toluene solution) dropwise.3-methoxybenzonitrile (4.29 g, 32.2 mmol) and AlCl₃ (1.575 g, 11.81mmol, 40 ml Toluene solution) were added to the above mixturesubsequently. The resulting mixture was stirred at RT for 1 h, thenheated at 110° C. for 6 hrs. The reaction mixture was cooled to RT, towhich aq. HCl (1 M, 13 ml) was added. The solution was then heated at80° C. for 1 h. The solution was cooled to RT, and the organic layer andwater layer were separated. The water layer was extracted with ethylacetate (3×50 mL). The combined organic layers were washed with waterand brine, and dried over sodium sulfate. The organic solvent was thenremoved under vacuum. The crude product was purified by silicachromatography with 0-40% ethyl acetate/heptane to give 1 (875 mg, 3.85mmol) in 36% yield. ESI MS (m/z, MH⁺): 227.9. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 3.74 (s, 3H) 6.00 (br. s., 2H) 6.44-6.56 (m, 1H)6.63 (dd, J=8.53, 1.00 Hz, 1H) 6.93-7.00 (m, 1H) 7.04-7.11 (m, 2H)7.14-7.31 (m, 2H) 7.37 (dd, J=8.03, 1.51 Hz, 1H).

A mixture of 1 (570 mg, 2.51 mmol) and ethyl 4-chloro-4-oxobutanoate(454 mg, 2.76 mmol) in DCM (20 ml) was heated at 60° C. for 1 h. Thereaction mixture was cooled and quenched with aq. 1 M NaOH (5 ml). Theorganic layer and water layer were separated. The water layer wasextracted with dichloromethane (3×15 ml). The combined organic layerswere washed with water and brine, and dried over sodium sulfate. Theorganic solvent was then removed under vacuum to give 2 (824 mg, 2.32mmol) in 92% yield. ESI MS (m/z, MH⁺): 355.4. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.25-1.48 (m, 3H) 2.73-3.01 (m, 4H) 3.88 (s, 3H)4.18 (q, J=7.07 Hz, 2H) 7.05-7.20 (m, 2H) 7.22-7.30 (m, 2H) 7.37-7.45(m, 1H) 7.53-7.64 (m, 2H) 8.64 (d, J=8.59 Hz, 1H) 10.90 (br. s., 1H).

A mixture of 2 (824 mg, 2.32 mmol) and sodium hydride (927 mg, 23.19mmol) in ethanol (20 ml) was heated at 80° C. for 2 h. The reactionmixture was cooled to RT and quenched with water (5 ml) then neutralizedwith aq. 1 M HCl (2 ml). The resulting solution was extracted with ethylacetate (3×10 mL). The combined organic layers were washed with waterand brine, and dried over sodium sulfate. The organic solvent was thenremoved under vacuum to give 3 (583 mg, 0.81 mmol) in 81% yield. ESI MS(m/z, MH⁺): 310.1. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 3.49-3.64 (m,2H) 3.83-3.89 (m, 3H) 6.83-6.96 (m, 2H) 7.00-7.28 (m, 4H) 7.37-7.56 (m,4H) 12.02-12.32 (m, 2H).

A solution of 3 (583 mg, 1.89 mmol) in POCl₃ (10 ml) was stirred at RTfor 2 h. POCl₃ was removed under vacuum, the resulting residue wasquenched with ethanol (20 ml). The solution was stirred at RT for 1 h,then ethanol was removed under vacuum. To the residue was addeddichloromethane (30 ml) and aq. 1 M NaOH (20 ml). Organic layer andwater layer were separated. The water layer was extracted withdichloromethane (2×20 ml). The combined organic layers were washed withwater, brine and dried over sodium sulfate. The organic solvent wasremoved under vacuum to give 4 (760 mg, 2.25 mmol) in 120% yield. ESI MS(m/z, MH⁺): 338.1. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.26 (t, J=7.07Hz, 3H) 3.44-3.64 (m, 2H) 3.85 (s, 3H) 4.17 (q, J=7.16 Hz, 2H) 6.85-6.93(m, 2H) 7.03 (ddd, J=8.46, 2.65, 1.01 Hz, 1H) 7.11 (ddd, J=8.21, 6.95,1.01 Hz, 1H) 7.17 (dd, J=8.21, 1.39 Hz, 1H) 7.32-7.39 (m, 1H) 7.40-7.52(m, 2H) 11.43 (br. s., 1H)

A solution of 4 (760 mg, 2.25 mmol) in POCl₃ (10 ml) was heated at 110°C. for 12 h. POCl₃ was removed under vacuum. To the residue was addeddichloromethane (20 ml) and aq. 1 M NaOH (20 ml). The organic layer andwater layer were separated. The water layer was extracted withdichloromethane (2×20 ml). The combined organic layers were washed withwater, brine and dried over sodium sulfate. The organic solvent wasremoved under vacuum to give 5 (650 mg, 1.83 mmol) in 97% yield. ESI MS(m/z, MH⁺): 355.4. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.25 (t, J=7.28Hz, 3H) 3.75 (d, J=1.00 Hz, 2H) 3.85 (s, 3H) 4.18 (q, J=7.03 Hz, 2H)6.78-6.92 (m, 2H) 6.97-7.13 (m, 1H) 7.39-7.60 (m, 3H) 7.76 (d, J=2.01Hz, 1H) 8.15 (d, J=8.53 Hz, 1H).

A mixture of 5 (650 mg, 1.83 mmol), triethylamine (3.14 g, 31.1 mmol),and Pd/C (10%, 194 mg, 1.827 mmol) in ethanol (20 ml) was stirred underH₂ (1 atm) at RT for 12 h. The reaction mixture was filtered to removePd/C. The organic solvent was removed under vacuum to give 6 (460 mg,1.43 mmol) in 78% yield. ESI MS (m/z, MH⁺): 321.5. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 1.20-1.26 (m, 3H) 1.44 (t, J=7.53 Hz, 9H) 3.12 (qd,J=7.28, 4.77 Hz, 6H) 3.65 (s, 2H) 3.79-3.93 (m, 3H) 4.12 (q, J=7.19 Hz,2H) 6.82-6.92 (m, 2H) 7.06 (ddd, J=8.53, 2.51, 1.00 Hz, 1H) 7.39-7.58(m, 3H) 7.72 (ddd, J=8.41, 6.65, 1.51 Hz, 1H) 8.19 (d, J=8.53 Hz, 1H)8.93 (s, 1H) 12.20 (br. s., 3H).

To a solution of 6 (400 mg, 1.245 mmol) in DCM (15 ml) was added BBr₃ (1M in DCM, 3.73 ml, 3.73 mmol) at −78° C. The reaction mixture was warmedto RT in 2 h. The mixture was cooled down to −78° C., and quenched withethanol. The organic solvent was removed under vacuum. To the resultingresidue was added ethyl acetate (15 ml) and water (15 ml). The organicand water layer were separated. The water layer was extracted with ethylacetate (3×15 ml). The combined organic layers were washed with water,brine and dried over sodium sulfate. The organic solvent was removedunder vacuum. The crude was purified by recrystallization fromdichloromethane to give 7 (282 mg, 0.92 mmol) in 74% yield. ESI MS (m/z,MH⁺): 308.2. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.11 (t, J=7.03 Hz, 3H)3.68 (s, 2H) 3.91-4.11 (m, 2H) 6.50-6.70 (m, 2H) 6.81-6.97 (m, 1H)7.30-7.48 (m, 2H) 7.51-7.63 (m, 1H) 7.78 (ddd, J=8.53, 7.03, 1.51 Hz,1H) 8.08 (d, J=8.03 Hz, 1H) 8.93 (s, 1H) 9.75 (br. s., 1H).

A mixture of 7 (20 mg, 0.065 mmol), benzyl bromide (16.69 mg, 0.098mmol) and cesium carbonate (42.4 mg, 0.13 mmol) in DMF (500 ul) wasstirred at RT for 12 hrs. The reaction mixture was filtered to removeinsoluble material. The crude product was purified by HPLC with 5% NH₄OHin 5-95% acetonitrile/water to give 8 (6.9 mg, 0.017 mmol) in 26.7%yield. ESI MS (m/z, MH⁺): 398.3. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm1.22 (t, J=7.03 Hz, 3H) 3.64 (s, 2H) 4.12 (q, J=7.03 Hz, 2H) 5.11 (s,2H) 6.76-7.02 (m, 2H) 7.13 (ddd, J=8.53, 2.51, 1.00 Hz, 1H) 7.31-7.56(m, 8H) 7.71 (ddd, J=8.28, 6.78, 2.01 Hz, 1H) 8.17 (d, J=8.53 Hz, 1H)8.92 (s, 1H).

A mixture of 8 (6.9 mg, 0.017 mmol), aq. 1 M LiOH (0.019 ml, 0.019 mmol)in Dioxane (0.5 ml) was stirred at RT for 12 hrs. The organic solventwas removed under vacuum to give 9 (6 mg, 0.016 mmol) in 92% yield aslithium salt. ESI MS (m/z, MH⁺): 370.2. ¹H NMR (400 MHz, METHANOL-d₄) 6ppm 3.42-3.60 (m, 2H) 5.07-5.19 (m, 2H) 6.94 (dt, J=7.53, 1.25 Hz, 1H)7.06 (dd, J=2.51, 1.51 Hz, 1H) 7.14 (ddd, J=8.53, 2.51, 1.00 Hz, 1H)7.25-7.42 (m, 3H) 7.42-7.53 (m, 2H) 7.70 (ddd, J=8.41, 4.64, 3.51 Hz,2H) 8.04 (d, J=8.03 Hz, 1H) 8.56 (s, 2H) 8.88 (s, 1H).

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 9 (4.08 mg,0.011 mmol), and DIC (2.74 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₃—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 10. TOF MS (ES⁻): 6495.

2. DD. Synthesis of siRNA Conjugated with X1017

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 9 (4.07 mg,0.011 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 11. TOF MS (ES⁻): 6510.

2. EE. Synthesis of siRNA Conjugated with X1022

A mixture of N-hydroxysuccinimide (2.48 mg, 0.022 mmol), 9 (4.06 mg,0.011 mmol), and DIC (2.72 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₅—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 12. TOF MS (ES⁻): 6524.

2. FF. Synthesis of siRNA Conjugated with X1024

A mixture of N-hydroxysuccinimide (2.5 mg, 0.0522 mmol), 1 (2.5 mg,0.013 mmol), and DIC (2.74 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₃—NH₂solution (2.0 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6315.

2. GG. Synthesis of siRNA Conjugated with X1026

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 1 (2.02 mg,0.011 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6327.

2. HH. Synthesis of siRNA Conjugated with X1025

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 1 (2.84 mg,0.01 mmol), and DIC (2.74 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₃—NH₂solution (2.0 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6390.

2. II. Synthesis of siRNA Conjugated with X1027

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 1 (2.84 mg,0.011 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6404.

2. JJ. Synthesis of siRNA Conjugated with X1028

A mixture of N-hydroxysuccinimide (2.48 mg, 0.022 mmol), 1 (2.90 mg,0.011 mmol), and DIC (2.72 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₅—NH₂solution (2 mg, 0.324 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 2. TOF MS (ES⁻): 6417.

2.KK. Synthesis of siRNA Conjugated with X1062

To AlCl₃ (7.88 g, 59.1 mmol) in Toluene (200 ml) was added aniline (5 g,53.7 mmol, 4.59 ml, in 50 ml Toluene) dropwise under N₂.3-bromobenzonitrile (29.3 g, 161 mmol) was added to the above mixturesubsequently. The resulting mixture was stirred at RT for 1 h, thenheated at 110° C. for 6 hrs. The reaction mixture was cooled to RT, towhich aq. HCl (1 M, 3 ml) was added. The solution was then heated at 80°C. for 1 h. The solution was cooled to RT, and the organic layer andwater layer were separated. The water layer was extracted with ethylacetate (3×100 mL). The combined organic layers were washed with waterand brine, and dried over sodium sulfate. The organic solvent was thenremoved under vacuum. The crude product was purified by silicachromatography with 0-40% ethyl acetate/heptane to give 1 (4.31 g, 15.6mmol) in 29% yield. ESI MS (m/z, MH⁺): 278.1 ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 6.16 (br. s., 2H) 6.64 (t, J=7.53 Hz, 1H) 6.76 (d,J=8.53 Hz, 1H) 7.29-7.49 (m, 3H) 7.56 (d, J=7.53 Hz, 1H) 7.67 (d, J=8.03Hz, 1H) 7.74-7.84 (m, 1H)

A mixture of 1 (1.02 g, 3.69 mmol) and ethyl 4-chloro-4-oxobutanoate(0.669 g, 4.06 mmol) in DCM (60 ml) was heated at 60° C. for 1 h. Thereaction mixture was cooled and quenched with aq. 1 M NaOH (15 ml). Theorganic layer and water layer were separated. The water layer wasextracted with dichloromethane (3×50 ml). The combined organic layerswere washed with water and brine, and dried over sodium sulfate. Theorganic solvent was then removed under vacuum to give 2 (1.44 g, 3.56mmol) in 96% yield. ESI MS (m/z, MH⁺): 406.2. ¹H NMR (400 MHz,Chloroform-d) δ 10.88 (s, 1H), 8.65 (d, J=8.4 Hz, 1H), 7.86 (d, J=1.8Hz, 1H), 7.74 (d, J=8.0 Hz, 1H), 7.65-7.51 (m, 3H), 7.39 (t, J=7.8 Hz,1H), 7.12 (t, J=7.7 Hz, 1H), 4.17 (q, J=7.1 Hz, 2H), 2.77 (dt, J=10.3,5.2 Hz, 4H), 1.27 (t, J=7.1 Hz, 4H).

A mixture of 2 (1.44 g, 3.56 mmol) and sodium hydride (1.425 g, 35.6mmol) in ethanol (20 ml) was heated at 80° C. for 2 h. The reactionmixture was cooled to RT and quenched with water (5 ml) then neutralizedwith aq. 1 M HCl (2 ml). The resulting solution was extracted with ethylacetate (3×50 mL). The combined organic layers were washed with waterand brine, and dried over sodium sulfate. The organic solvent was thenremoved under vacuum to give 3 (1.2 g, 3.63 mmol) in 94% yield. ESI MS(m/z, MH⁺): 360.2. ¹H NMR (400 MHz, Chloroform-d) δ 11.71-11.63 (m, 1H),11.55-11.42 (m, 3H), 11.37 (d, J=8.3 Hz, 1H), 11.30-11.22 (m, 1H), 11.12(td, J=7.6, 7.0, 1.2 Hz, 1H), 11.00 (dd, J=8.2, 1.4 Hz, 1H), 7.33 (d,J=3.5 Hz, 2H), 4.87-4.82 (m, 2H).

A solution of 3 (1.2 g, 1.89 mmol) in POCl₃ (15 ml) was stirred at RTfor 2 h. POCl₃ was removed under vacuum, the resulting residue wasquenched with ethanol (50 ml). The solution was stirred at RT for 2 h,then ethanol was removed under vacuum. To the residue was addeddichloromethane (50 ml) and aq. 1 M NaOH (20 ml). Organic layer andwater layer were separated. The water layer was extracted withdichloromethane (2×50 ml). The combined organic layers were washed withwater, brine and dried over sodium sulfate. The organic solvent wasremoved under vacuum to give 4 (1.76 mg, 4.56 mmol) in 136% yield. ESIMS (m/z, MH⁺): 388.2. ¹H NMR (400 MHz, Chloroform-d) δ 7.65 (dt, J=8.2,1.4 Hz, 1H), 7.54-7.46 (m, 2H), 7.45-7.37 (m, 2H), 7.27 (dt, J=7.8, 1.5Hz, 1H), 7.17-7.04 (m, 2H), 4.18-4.16 (m, 2H), 3.50 (d, J=1.5 Hz, 2H),1.27 (h, J=3.7 Hz, 3H).

A solution of 4 (1.76 g, 4.56 mmol) in POCl₃ (10 ml) was heated at 110°C. for 12 h. POCl₃ was removed under vacuum. To the residue was addeddichloromethane (50 ml) and aq. 1 M NaOH (20 ml). The organic layer andwater layer were separated. The water layer was extracted withdichloromethane (2×50 ml). The combined organic layers were washed withwater, brine and dried over sodium sulfate. The organic solvent wasremoved under vacuum to give 5 (440 mg, 1.09 mmol) in 32.5% yield. ESIMS (m/z, MH⁺): 406.0. ¹H NMR (400 MHz, Chloroform-d) δ 8.14-8.05 (m,1H), 7.76 (ddd, J=8.4, 6.9, 1.4 Hz, 1H), 7.69 (ddd, J=8.0, 1.9, 1.0 Hz,1H), 7.53-7.41 (m, 3H), 7.36 (dd, J=8.3, 1.3 Hz, 1H), 7.26 (dt, J=7.7,1.3 Hz, 1H), 4.19 (q, J=7.1 Hz, 2H), 3.72 (s, 2H), 1.27 (t, J=7.1 Hz,3H).

A mixture of 5 (50 mg, 0.124 mmol), Zinc power (40.4 mg, 0.618 mmol) inTFA (1 ml) was heated at 40° C. for 12 h. The reaction mixture wasquenched with NaOH (1M, 1 ml). The organic layer was extracted withdichloromethane (2×10 ml). The combined organic layers were washed withwater, brine and dried over sodium sulfate. The organic solvent wasremoved under vacuum. The crude product was purified by silica flashchromotograph with elute 0-50% EtOAc/Heptane to give 6 (39 mg, 0.105mmol) in 85% yield. ESI MS (m/z, MH⁺): 372.1. ¹H NMR (400 MHz,CHLOROFORM-d) ppm 1.24 (t, J=7.15 Hz, 3H) 3.63 (s, 2H) 4.02-4.27 (m, 2H)7.12-7.33 (m, 1H) 7.37-7.61 (m, 4H) 7.61-7.71 (m, 1H) 7.75 (t, J=1.51Hz, 1H) 8.21 (d, J=8.53 Hz, 1H) 8.94 (s, 1H).

A mixture of4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ethyl)-1,3-dioxolan-2-one)(255 mg, 1.05 mmol), 6 (39 mg, 0.105 mmol), (Brettphos)paddadium(II)phenethylamine chloride (4.21 mg, 5.27 umol) and 1M aqueous K₃PO₄ (421uL, 0.421 mmol) in 2-Me THF (500 uL) and DMF (500 uL) was heated at 80°C. for 12 h. The reaction mixture was filtered to remove insolublematerial. The organic solvent was removed under vacuum. The crude waspurified by HPLC with 5% TFA in 5-95% acetonitrile/water to give 7 (15mg, 0.04 mmol) in 37.5% yield. ESI MS (m/z, MH⁺): 380.4. ¹H NMR (400MHz, CHLOROFORM-d) ppm 1.22 (td, J=7.15, 2.26 Hz, 3H) 1.73-1.92 (m, 2H)2.72-2.98 (m, 2H) 3.49 (dd, J=11.04, 7.53 Hz, 1H) 3.56-3.84 (m, 4H)4.04-4.20 (m, 2H) 7.15 (d, J=7.53 Hz, 1H) 7.13 (d, J=8.78 Hz, 1H) 7.37(d, J=7.53 Hz, 1H) 7.43-7.56 (m, 3H) 7.74 (br. s., 1H) 8.22 (br. s., 1H)8.94 (br. s., 1H).

A mixture of 7 (15 mg, 0.04 mmol) and aq. 1 M LiOH (87 uL ml, 0.087mmol) in dioxane (200 ul) was stirred at RT for 12 hrs. The organicsolvent was removed under vacuum to give 8 (14.17 mg, 0.04 mmol) in 100%yield as lithium salt. ESI MS (m/z, MH⁺): 352.4. ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 1.62-1.80 (m, 1H) 1.80-1.98 (m, 1H) 2.78 (ddd,J=13.33, 6.76, 3.16 Hz, 1H) 2.84-2.99 (m, 1H) 3.46-3.55 (m, 3H)3.55-3.68 (m, 3H) 6.98-7.25 (m, 2H) 7.31-7.59 (m, 4H) 7.74 (ddd, J=8.40,6.38, 1.89 Hz, 1H) 8.06 (d, J=8.34 Hz, 1H) 8.86 (s, 1H).

A mixture of 8 (14 mg, 0.039 mmol), TBDMSCI and imidazole (43.6 mg,0.641 mmol) in DMF (4 ml) was stirred at RT for 24 hrs. The reactionmixture was quenched with water (1 ml). The organic layer was extractedwith ethylestate (3×5 ml). The combined organic layers were washed withwater, brine and dried over sodium sulfate. The organic solvent wasremoved under vacuum. The crude product was purified by silica flashchromotograph with elute 0-50% EtOAc/Heptane to give 9 (15.9 mg, 0.025mmol) in 63.2% yield. ESI MS (m/z, MH⁺): 580.6. ¹H NMR (400 MHz,METHANOL-d₄) δ ppm-0.12-0.13 (m, 8H) 0.69-0.98 (m, 12H) 1.17-1.36 (m,5H) 2.60-2.90 (m, 2H) 3.38-3.62 (m, 6H) 3.73 (d, J=4.55 Hz, 1H)7.02-7.15 (m, 2H) 7.24-7.38 (m, 1H) 7.39-7.47 (m, 3H) 7.64-7.74 (m, 1H)7.98-8.06 (m, 1H) 8.68-8.89 (m, 1H).

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 9 (6.26 mg,0.011 mmol), and DIC (2.74 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 hrs. 10 uL of reaction mixture was diluted with 190 uLDMSO. To the resulting solution was added a freshly ss-siRNA-(CH₂)₃—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 10. TOF MS (ES⁻): 6478.

2.LL. Synthesis of siRNA Conjugated with X1063

A mixture of N-hydroxysuccinimide (2.49 mg, 0.022 mmol), 9 (6.26 mg,0.011 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₄—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 11. TOF MS (ES⁻): 6492.

2.MM. Synthesis of siRNA Conjugated with X1064

A mixture of N-hydroxysuccinimide (2.48 mg, 0.022 mmol), 9 (6.26 mg,0.011 mmol), and DIC (2.72 mg, 0.022 mmol) in DMSO (200 uL) was stirredat RT for 12 h. 10 uL of reaction mixture was diluted with 190 uL DMSO.To the resulting solution was added a freshly ss-siRNA-(CH₂)₅—NH₂solution (2 mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reactionmixture was vortexed and sat at RT for 2 h. The crude product waspurified by HPLC with 10-40% 100 mM triethylammonium acetate inacetonitrile/water to afford 12. TOF MS (ES⁻): 6506.

Example 3 PAZ Ligand Conjugate 3′ End Cap Activity In Vitro and In Vivoand Structure

PAZ ligand conjugates were studied as a part of a 7-mer RNA conjugatestructure in X-ray crystal and NMR structural studies (data not shown).

RNA interference activity of duplexes comprising various 3′ end caps wasalso analyzed; in vitro and in vivo potency was studied, as shown inExample 3A (in vitro data) and 3B (in vivo data).

Example 3a In Vitro Potency of RNAi Agents Comprising a 3′ End Cap (PAZLigand)

Potency of HAMP siRNA—PAZ Ligand Conjugates is studied.

Hepcidin mRNA down regulation in HuH-7 cells is studied at 3 doses.

Two test sequences: hs_HAMP_400 and 402

Parent stem format: A106S42 (2′-OMe chemistry

19 PAZ ligands on guide (antisense) strand+/− ribitol spacer, +/−MOEclamp (wherein the MOE clamp is a 2′-MOE modification on each of the twolast base-pairing nucleotides on each strand counting from 5′ to 3′).

In vitro dose-response in Huh-7 cells is determined.

Results are shown in FIGS. 5A and 5B and 7 and in Table 5, below.

FIGS. 5A and 5B show the in vitro RNA interference or KD (knockdown)mediated by various RNAi agents comprising a 3′ end cap: BP (biphenyl),C6, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063,X064, X065, X066, X067, X068, and X069 on the guide (antisense) strand.Two sequences were tested (hs_HAMP_400 and _402), where 400 is asequence beginning at position 400 of the human HAMP (Hepcidin) gene and402 is an overlapping sequence beginning at position 402

These were tested with (Rib) and without (−) a ribitol spacer; and with(MOE) and without (−) a 2′-MOE clamp (as diagrammed in FIGS. 6A and 6B).Various hs_HAMP 400 and 402 are depicted in FIG. 6A (Guide or antisensestrand) and 6B (corresponding Sense strand).

The data are provided in FIGS. 5A and 5B. The circled data points inFIGS. 4A and 4B represent the most potent format for hs_HAMP_400 and themost potent format for the hs_HAMP_402.

Additional data is provided in Table 5, below. This Table indicates theNickname (“Oligo Identifier”) for the 3′ end cap, and the DMT, Succinateand Carboxylate variants thereof; the Carboxylate Kd; the KD (knockdown)mediated by a Hepcidin RNAi agent comprising the 3′ end cap (format:S402+ribitol+MOE clamp) at 5 nM in vitro; and the approximate (approx.)IC50

TABLE 6 Oligo Hepcidin Hepcidin Identifier KD at 5 nM (approx.)(Nickname) in vitro (%) IC50 μM BP 68 2.3 (biphenyl) X027 57 3.3 X038 622.6 X050 61 4.0 X051 61 3.4 X052 64 3.0 X058 68 2.7 X059 55 4.1 X060 653.5 X061 61 3.0 X062 63 3.5 X063 56 3.7 X064 60 3.1 X065 66 3.0 X066 494.9 X067 66 2.5 X068 63 3.0 X069 81 1.5 C6 66 2.6

FIG. 7 shows the residual gene activity (wherein residual geneactivity=100%−KD) of Hepcidin mm-reporter levels at 72 hours in COS1cells after various doses of RNAi agents comprising a 3′ end cap, at arange from 1.57 nM to 15 nM. The format of the strands is indicated. The3′ end of the sense strand terminates in a 2′ MOE-clamp—ribp (ribitolspacer)—C6. The 3′ end of the antisense strand terminates in a 2′MOE-clamp—ribp (ribitol spacer)—ligand, wherein the ligands used were 3′end caps (X027, X058, X067, etc.).

These data show the efficacy of RNAi agent comprising a 3′ end cap whichis BP (biphenyl), C6, X027, X038, X050, X051, X052, X058, X059, X060,X061, X062, X063, X064, X065, X066, X067, X068, or X069.

Example 3B In Vivo Potency of RNAi Agents Comprising a 3′ End Cap (PAZLigand)

Example 3A showed the in vitro potency of various RNAi agents comprisinga 3′ end cap.

Example 3B shows the in vivo potency of various RNAi agents comprising a3′ end cap.

These in vivo experiments used these parameters:

Mice (n=5/group) injected via IV bolus (tail vein): LNP569

-   -   PBS    -   LNP569-Hamp254-X052 (SL52-49CE)-3 mg/kg    -   LNP569-Hamp254-X058 (IL54-43-XE)-3 mg/kg    -   LNP569-Hamp254-X067 (YL55-48RE)-3 mg/kg    -   LNP569-Hamp254-X038 (CL51-55IE)-3 mg/kg    -   LNP569-Hamp254-X069 (GA35-24OF)-3 mg/kg    -   LNP569-Hamp254-X027 (ML59-39NE)-3 mg/kg    -   LNP569-Hamp254-C6 control-3 mg/kg        LNP569 is a lipid nanoparticle preparation of the RNAi agent.

Two timepoints-48 and 168 hrs post-injection (both 3 mg/kg).

Assess hepcidin knockdown in liver (mRNA-qPCR)

Key Questions are Asked:

Are PAZ domain ligands active in vivo?(48 hour timepoint).Do PAZ domain ligands provide benefit for duration of knockdown?(168hour timepoint).

The results are shown in FIGS. 8A and 8B.

FIGS. 8A and 8B show that in both the ABI Hamp1 Taqman assay (FIG. 8A)and the Hamp1 specific Taqman Assay (FIG. 8B) all of the RNAi agentswere able to mediate Hepcidin knockdown in vivo at 48 hours post-dose,with a 1×3 mg/kg dose. 3′ end caps used were: X052, X058, X067, X038,X069, and X027, with C6 as a control.

The finding that RNAi agents with 3′ end caps of X052, X058, X067, X038,X069, X027, or C6 were still able to mediate RNA interference at 48hours indicates that the 3′ end caps protect the RNAi agents againstdegradation or digestion (e.g., by nucleases in the serum).

FIG. 9A shows that in the Hamp1 specific Taqman assay, the duplexcomprising the X058 3′ end cap was still able to mediate RNAinterference (measured by Hepcidin knockdown) at 168 hours post-dose invivo. Thus, >50% knockdown was observed in mice after 7 days with asingle dose.

Without being bound by any particular theory, this disclosure notes thatthe increased potency and duration of knockdown mediated by RNAi agentswith a X058 3′ end cap may be due to the increased association of X058with Ago2. FIG. 9B shows the X058 and C6 Ago2 Pulldown experiment usingHepcidin 18-mer oligonucleotides.

Briefly, antibodies to Ago2 were used to pull down Ago2 from cells 72after dosage with RNAi agents comprising either a X058 or C6 3′ end cap,or a non-targeting (NT) control RNAi agent. Analysis was then performedto determine levels of RNAi agents, as shown. FIG. 9B shows that, after72 hrs, much more RNAi agent with X058 was associated with Ago2 than theRNAi agent with C6.

Thus, these data show that:

HAMP 18-mer (254) siRNAs with X038, X052, X058, X067, or X069 PAZligands on guide strand are active in vivo.

X058 shows convincing increased potency and duration of knockdown.

Additional In Vivo Testing.

An additional in vivo testing was done with different chemical formats:(A160_S38, S42, S45 & A161_S38, S42, S45).

In the Antisense Strand:

A160_→F in position 2 and ribC6 overhangA161_→F in position 2, 5, 6, 7 and ribC6 overhangIn the sense strand:_S38→C6 overhang_S42→ribC6 overhang_S45→BP overhang

Parameters used in this experiment were:

Mice (n=5/group) injected via IV bolus (tail vein): LNP569

-   -   PBS    -   LNP569-Hamp254 A160_S38-3 mg/kg    -   LNP569-Hamp254 A160_S42-3 mg/kg    -   LNP569-Hamp254 A160_S45-3 mg/kg    -   LNP569-Hamp254 A161_S38-3 mg/kg    -   LNP569-Hamp254 A161_S42-3 mg/kg    -   LNP569-Hamp254 A161_S45-3 mg/kg

48 Hour Timepoint.

Assess Hepcidin Knockdown in Liver (mRNA-qPCR)

The results are shown in FIG. 10. FIG. 10 shows the In vivo comparisonof A160 & A161 format [various passenger (sense) strand overhangs]. Thisexperiment was done 48 hours post-dose, with a 1×3 mg/kg dose.

These data show the in vivo efficacy of RNAi agents comprising any ofseveral 3′ end caps: BP, C6, X052, X058, X067, X038, X069, X027,

Example 4 Additional Studies Showing Efficacy of 3′ End Caps

Additional studies are performed using RNAi agents comprising a 3′ endcap.

FIG. 16A shows the efficacy of RNAi agents wherein the 3′ end cap isX109, X110, X111, X112, X113, X058 or C6. HuR is the target. Doses usedare: 1 nM, 0.25 nM, 0.62 nM, and 0.16 nM. RNAi agents comprising any ofthe 3′ end caps were able to mediate RNA interference, particularly atthe highest doses used.

In particular, HuR-PAZ ligands X110, X111 and X112 appear to be similarin potency as X058.

Table 6, below, provides additional data showing the efficacy of 18-merformat RNAi agents with various 3′ end caps: X059, X050, X061, X051,X027, X062, X060, C6 (X003), X068, X065, X069, X097, X066, X098, X052,X063, BP (X014), X038, X067, X058, X064, and ribprib (X025).

TABLE 7 Efficacy of RNAi agents with a 3' end cap to ELAVL1/HuR in cellsin vitro AV 5.04 9.95 19.85 SD Nickname siTrack siRNA % residual mRNA(qRT-PCR) 5.04 9.95 19.85 untreated-HB untreated 100.54 10.76av_PSAT6-EYFP- eYFP neg. control 1 110.06 104.72 101.98 6.52 13.38 11.84N1_471_A25S27 av_PNAS-280_1_A25S27 eYFP neg. control 2 102.15 96.1698.87 15.02 6.60 9.41 hs_ELAVL1_1186_MAN_S42 18-mer siRNA with X05945.17 20.58 10.24 2.84 0.63 0.79 hs_ELAVL1_1186_MAN_S42 18-mer siRNAwith X050 26.69 11.48 6.92 0.99 2.40 1.41 hs_ELAVL1_1186_MAN_S42 18-mersiRNA with X061 26.11 11.49 6.25 5.81 0.99 0.74 hs_ELAVL1_1186_MAN_S4218-mer siRNA with X051 25.35 10.80 6.88 3.28 0.61 0.86hs_ELAVL1_1186_MAN_S42 18-mer siRNA with X027 24.54 11.67 6.17 2.90 1.381.03 hs_ELAVL1_1186_MAN_S42 18-mer siRNA with X062 24.35 11.68 5.66 2.881.46 1.17 hs_ELAVL1_1186_MAN_S42 18-mer siRNA with X060 23.86 9.27 5.621.10 0.86 0.76 hs_ELAVL1_1186_A106_S42 18-mer siRNA with C6 (X003) 22.429.77 5.65 1.90 1.60 1.31 hs_ELAVL1_1186_MAN_S42 18-mer siRNA with X06822.40 10.77 5.89 2.25 1.92 1.31 hs_ELAVL1_1186_MAN_S42 18-mer siRNA withX065 22.24 10.50 5.20 3.44 1.26 0.96 hs_ELAVL1_1186_MAN_S42 18-mer siRNAwith X069 21.93 9.57 6.13 6.25 1.11 1.57 hs_ELAVL1_1186_MAN_S42 18-mersiRNA with X097 21.26 9.83 6.29 3.45 1.98 1.27 hs_ELAVL1_1186_MAN_S4218-mer siRNA with X066 21.12 10.25 5.77 2.04 1.14 0.43hs_ELAVL1_1186_MAN_S42 18-mer siRNA with X098 21.06 9.94 6.15 4.39 2.161.29 hs_ELAVL1_1186_MAN_S42 18-mer siRNA with X052 21.02 8.32 6.75 1.411.29 0.93 hs_ELAVL1_1186_MAN_S42 18-mer siRNA with X063 20.53 10.91 5.613.01 0.86 0.18 hs_ELAVL1_1186_A324_S42 18-mer siRNA with BP (X014) 20.389.37 6.19 2.56 1.37 0.67 hs_ELAVL1_1186_A27_S30 19-mer pos. control19.90 8.03 5.40 1.40 0.98 0.37 hs_ELAVL1_1186_MAN_S42 18-mer siRNA withX038 19.80 10.60 5.26 2.52 0.75 0.75 hs_ELAVL1_1186_MAN_S42 18-mer siRNAwith X067 19.07 11.38 5.82 2.02 3.00 0.94 hs_ELAVL1_1186_MAN_S42 18-mersiRNA with X058 18.40 10.36 6.23 2.70 2.78 0.42 hs_ELAVL1_1186_MAN_S4218-mer siRNA with X064 18.33 9.84 6.49 3.03 0.45 1.67hs_ELAVL1_1186_MAN_S42 18-mer siRNA with ribprib 17.10 9.78 5.75 3.691.38 0.75 (X025) hs_ELAVL1_1186_A22_S26 21-mer pos. control 16.68 8.615.15 2.39 1.17 0.86

This table provides: the nickname of the RNAi agent (column 1); thelength and the 3′ end cap used and identification of controls (column2); % residual mRNA level, as determined by qRT-PCR, at doses of 5.04,9.95, and 19.85 nM (columns 3-5); standard deviation (SD) (columns 6-8).HuR is normalized to Cyc. 5.04, 9.95, 19.85, 5.04, 9.95, 19.85 representdosing (nM).

The knockdown (RNA interference activity) can be readily calculated bysubtracting the % residual mRNA from 100%. Thus, the final line showsthat the 21-mer pos. (positive) control exhibits 16.68% residual mRNA,indicating 83.32% knockdown.

These data show the efficacy of RNAi agent comprising a first and asecond strand, wherein the 3′ end of the first and/or second strandterminates in a phosphate and further comprises in 5′ to 3′ order: aspacer (e.g., ritbitol), a phosphate, and a 3′ end cap.

These data show that efficacious RNAi agents can be constructed whereinthe 3′ end is BP (X014), C6 (X003), ribprib (X025), X027, X038, X050,X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067,X068, X069, X097, or X098.

Example 5 Efficacy of RNAi Agents Comprising a C8 or C10 3′ End Cap

RNAi agents comprising a C8 or C10 3′ end cap are tested.

19-mer SSB siRNA with C8 or C10 overhang are tested.

FIGS. 18A and 18B show the results, while FIG. 17C diagrams the various3′ end caps used.

RNAi agents comprising a C8 or C10 3′ end cap were able to mediate RNAinterference.

19-mer SSB siRNA with C10 overhang gives less potent mRNA downregulationat 3d than 21-mer positive control, but better duration of effect.

Thus, the C10 modified 19mer siRNA is not giving the same maximum targetknock-down at an early timepoint (day 3), but holds up longer (morepotent knockdown at day 10). The C10 3′ end cap thus provides a superiorduration of action.

Experimental data shows that efficacious RNAi agents can be constructedcomprising a 3′ end cap which is C6, C8 or C10. In addition, the C10 3′end cap has the advantage of increased duration of activity in vivo.

Example 6 RNAi Agents Comprising Phosphorothioate and/or RNA, DNA,2′-MOE, 2′-F, or LNA CLAMP

Variants a RNAi agent to F7 (Factor VII) are prepared.

These variants comprise, as examples, a 3′ end cap which isphosphorothioate-C3 (PS-C3), and/or a clamp. In the clamp, the last twobase-pairing nt counting from 5′ to 3′ are modified.

Results are shown in FIG. 20A-E.

FIG. 20 shows the efficacy of 3′ end caps and clamps comprising variousmodifications. FIGS. 20A and B show the efficacy of RNAi agentscomprising a 3′ end cap of phosphorothioate-C3 (PS-C3). FIGS. 20 C, Dand E show the efficacy of RNAi agents comprising a 2′ clamp, whereinthe last two base-pairing nt counting from 5′ to 3′ are RNA, DNA,2′-MOE, 2′-F, or LNA.

For the RNAi agents in FIGS. 20D and 20E, all the tested RNAi agentswere efficacious. It is noted that the percentages do not representknockdown, but knockdown relative to other RNAi agents. 100%, forexample, represents the average knockdown of all antisense strands ofthese efficacious RNAi agents.

These data thus show that efficacious RNAi agents can be constructedwhich comprise a RNA, DNA, 2′-MOE, 2′-F, or LNA, and in which themodified internucleoside linker is a phosphorothioate.

Example 7 RNAi Agents Comprising a C3, C4, or C5 Linker in the 3′ EndCap

This Example shows efficacy of RNAi agents comprising a 3′ end cap whichis: X109, X110, X111, X112, X113, X1009, X1010, X1024 or X1025 (FIG.23A); X1011, X1012, X1013, X058, X1015, X1016, X1017, X1026, X1027 (FIG.23B): or X1018; X1019, X1020, X1021, X1022 or X1028 (FIG. 23C).

The 3′ end caps shown in FIG. 20A comprise a C3; the 3′ end caps in FIG.20B comprise a C4; and the 3′ end caps in FIG. 20C comprise a C5. Thesedata show that RNAi agents comprising any of these 3′ end caps iseffacious.

A HuR RNAi agent is used.

In these experiments, Huh-7 cells are transfected using RNAiMax in a96-well plate format. RNA is isolated 48 hours post-transfection. HuRmRNA is normalized to PPIA endogenous control. RNAi agent concentrationsof 3, 10 and 30 pM are chosen based on IC50 data of the PAZ ligands (3′end caps) previously analyzed. For the X109 to X113 data, an average oftwo previous data sets is provided.

In general, length of the linker within the 3′ end cap does notsignificantly affect potency of any of the 3′ end caps.

In separate but related experiments, IC50 data was determined forseveral 3′ end caps using HuR RNAi agents in Huh-7 cells. Data pointsfor two separate studies are shown below:

siRNA 3′ pM IC50 pM IC50 end cap study#1 study#2 X058 5.85 12.78 X1093.47 3.85 X110 1.50 6.42 X111 1.21 3.63 X112 0.72 2.38 X113 2.71 4.55

These data show that RNAi agents comprising a 3′ end cap which is X058,X109, X110, X111, X112 or X113 are each efficacious.

Example 8 Additional RNAi Agents Comprising a C3, C4, or C5 Linker inthe 3′ End Cap

This example shows the efficacy of various RNAi agents comprising a 3′end cap which is: X110, X1012, X1018, X111, X1013, X112, X058, X1019,X1025, X1027, or X1028. This various 3′ end caps (illustrated inTable 1) vary in the length of the linker (C3, C4 or C5) between R₂ andthe head group.

As a note of clarification, this disclosure notes that the terms “C3”[—(CH₂)₃—], “C4” [—(CH₂)₄—], and “C5” [—(CH₂)₅-] are generally usedherein to designate spacers, similar terms (C3, C4, C5 “linkers”) arealso used to designate a portion of a 3′ end cap. In FIG. 13, thedifferent linkers are used to differentiate portions of various 3′ endcaps. It is also noted that the term “C3” is used to designate a C3 3′end cap (e.g., FIG. 15A), a C3 spacer (FIG. 21), and a C3 linker (FIG.13).

The target gene for this example is HuR. Huh-7 cells are transfectedusing RNAiMax transfection reagent. 24 well plates are seeded with40,000 cells per well. “Reverse transfection” with 1 nM RNAi agent/wellis done, followed by incubation for approximately 18 hours. Duplicateplates are set up using one for RNA extraction and the other forduration. Transfection media is replaced with fresh growth media (noRNAi agent) and cells are incubated for an additional 2 days before RNAisolation or split for duration experiments.

Cells are split on days 3 and 7 post-transfection. RNA is isolated atdays 3, 7 and 10 post-transfection for HuR mRNA analysis.

Results are shown in FIGS. 24A and 24B. The control (NTC) is a mFVII21-mer RNAi agent. In FIG. 24A, ligand LME844 (X110, X1012 and X1018),the linker length does not appear to alter the duration of activity. Forligand PKF027-895 (X111 and X1013), the shorter linker (C3) and the C4linker are not significantly different.

In FIG. 24B, for ligand LP1230 (X1025, X1027 and X1028), the duration ofthe C3 linker is better than the longer linkers. There is evidence ofthis as early as Day 7 post-transfection.

For ligand LKS871 (X112, X058 and X1019), the longer linker appears tohave slightly better activity at the later time point and the “trend” isthere at Day 7, as well, although the error bars overlaps and it isprobably not significant. The X058 activity at Day 10 is about 15% lessthan demonstrated in a previous duration study, but there will be studyto study variability for these types of analyses.

These data show that RNAi agents comprising a 3′ end cap which is X112,X058, X1019, X1025, X1027, or X1028 are each efficacious.

Example 9 Efficacy of Additional 3′ End Caps

The 3′ end caps X1062, X1063 and X1064 were each found to be efficaciouswhen used on RNAi agents. For example, these were effective on HuRsiRNAs, wherein the HuR siRNAs were 18-mers as described herein, whereinthe 3′ end of each strand terminates in a phosphate and furthercomprises, in 5′ to 3′ direction, a spacer which is ribitol, a secondphosphate, and a 3′ end cap which is X1062, X1063 or X1064. Huh7 cellswere transfected with siRNAs using RNAi Max transfection reagent;24-well plates were seeded with 40,000 cells per well; reversetransfection was performed with 1 nM siRNA per well, and cells wereincubated for about 18 hours. Transfection medium was replaced (withoutsiRNA), and cells were incubated for an additional 2 days before RNAisolation or split for seeding. Cells were split on days 3 and 7 posttransfection for duration time points. RNA was isolated at days 3, 7 and10 post-transfection for HuR mRNA analysis. HuR siRNAs with 3′ end capswhich were X1062 demonstrated efficacy (knockdown) of 89.0, 77.9 and32.7% after 3, 7 or 10 days. HuR siRNAs with 3′ end caps which wereX1063 and X1064 showed 89.6, 81.5, and 43.7%; and 67.0, 30.9 and 0.0%,respectively, after 3, 7 and 10 days.

Embodiments

-   -   1. A compound of formula Ia:

-   -   -   in which:        -   X is H; OH, wherein the hydroxyl group can optionally be            functionalized as succinate or attached to a solid support;            ODMT; carboxylic acid; the 3′ end of a strand of a RNAi            agent; or the 3′ end of a molecule comprising a strand of a            RNAi agent, wherein the 3′ end of the strand terminates in a            phosphate or modified internucleoside linker and further            comprises in 5′ to 3′ order: a spacer, and a second            phosphate or modified internucleoside linker;        -   Y is CH or N;        -   m is 0 or 1;        -   p is 1, 2 or 3;        -   R₃ is hydrogen, 2-(hydroxy-methyl)-benzyl,            3-(hydroxy-methyl)-benzyl, succinate, or a solid support;            -   wherein the (CH₂)_(m)—O—R₃ moiety is attached to the                phenyl ring at position 3 or 4;        -   R₄ is hydrogen;        -   R₅ is hydrogen; or        -   R₄ and R₅, together with the phenyl rings to which R₄ and R₅            are attached, form 6H-benzo[c]chromene.

    -   2. The compound of embodiment 1 selected from:

-   -   3. A compound of formula Ib:

-   -   -   in which:        -   X is H; OH, wherein the hydroxyl group can optionally be            functionalized as succinate or attached to a solid support;            ODMT; carboxylic acid; the 3′ end of a strand of a RNAi            agent; or the 3′ end of a molecule comprising a strand of a            RNAi agent, wherein the 3′ end of the strand terminates in a            phosphate or modified internucleoside linker and further            comprises in 5′ to 3′ order: a spacer, and a second            phosphate or modified internucleoside linker;        -   q is 0, 1 or 2;        -   R₆ is phenyl which is unsubstituted or substituted with a            group selected from benzoxy and 3,4-dihydroxybutyl;        -   R₇ is hydrogen or hydroxy-ethyl, wherein if R₇ is            hydroxy-ethyl, the hydroxyl can be optionally functionalized            as succinate or attached to a solid support;        -   R₈ is hydrogen or methoxy; Y₁ is CH or N; and        -   Y₂ is N or CR₉; wherein R₉ is selected from hydrogen and            methyl.

    -   4. The compound of embodiment 3 selected from:

-   -   5. A compound selected from:

-   -   -   In which:        -   X is H; OH, wherein the hydroxyl group can optionally be            functionalized as succinate or attached to a solid support;            ODMT; carboxylic acid; the 3′ end of a strand of a RNAi            agent; or the 3′ end of a molecule comprising a strand of a            RNAi agent, wherein the 3′ end of the strand terminates in a            phosphate or modified internucleoside linker and further            comprises in 5′ to 3′ order: a spacer, and a second            phosphate or modified internucleoside linker, and        -   q is selected from 1 and 2.

    -   6. A method for capping the 3′ end of a strand of an RNAi agent        comprising a method of the steps of:        -   reacting the RNAi agent with a compound selected from:

-   -   -   In which:        -   X is selected from H, OH, ODMT and carboxylate, wherein the            hydroxyl group can be optionally functionalized as succinate            or attached to a solid support;            -   Using solid-phase synthesis methods to replace X with a                strand of a RNAi agent; or        -   Constructing a RNAi agent strand on a solid support;        -   Reacting the strand with the compound, and        -   Cleaving the RNAi agent strand from the solid support.

    -   7. A compound of formula Ia, wherein X is H, OH, ODMT or        carboxylate, wherein the hydroxyl group can be optionally        functionalized as succinate or attached to a solid support; and        R₃ is hydrogen, 2-(hydroxy-methyl)-benzyl,        3-(hydroxy-methyl)-benzyl, succinate, or a solid support.

    -   8. A composition comprising a RNAi agent comprising a first        strand and a second strand, wherein the 3′-terminus of at least        one strand comprises a 3′ end cap, wherein the 3′ end cap is a        compound of any of embodiments 1 to 6, wherein X is the first or        second strand.

    -   9. The composition of embodiment 8, wherein the first and/or        second strands of the RNAi agent are no more than about 49        nucleotides long.

    -   10. The composition of embodiment 8, wherein the first and/or        second strands of the RNAi agent are no more than about 30        nucleotides long.

    -   11. The composition of embodiment 8, wherein the first and/or        second strand are 18 or 19 nucleotides long.

    -   12. The composition of embodiment 8, wherein the first strand is        the anti-sense strand and is 18 or 19 nucleotides long.

    -   13. The composition of embodiment 8, wherein the RNAi agent has        1 or 2 blunt-ends.

    -   14. The composition of embodiment 8, wherein the RNAi agent        comprises an overhang on at least one 5′ end or 3′ end.

    -   15. The composition of embodiment 8, wherein the RNAi agent        comprises a 1 to 6 nucleotide overhang on at least one 5′ end or        3′ end.

    -   16. The composition of embodiment 8, wherein the RNAi agent        comprises a spacer.

    -   17. The composition of embodiment 16, wherein the spacer is a        ribitol.

    -   18. The composition of embodiment 16, wherein the spacer is a        ribitol, 2′-deoxy-ribitol, diribitol, 2′-methoxyethoxy-ribitol        (ribitol with 2′-MOE), C3, C4, C5, C6, or        4-methoxybutane-1,3-diol.

    -   19. The composition of embodiment 8, wherein at least one        nucleotide of the RNAi agent is modified.

    -   20. The composition of embodiment 19, wherein said at least one        modified nucleotide is selected from among 2′        alkoxyribonucleotide, 2′ alkoxyalkoxy ribonucleotide, or        2′-fluoro ribonucleotide.

    -   21. The composition of embodiment 19, wherein said at least one        modified nucleotide is selected from 2′-OMe, 2′-MOE and 2′-H.

    -   22. The composition of embodiment 8, wherein one or more        nucleotides is modified or is DNA or is replaced by a peptide        nucleic acid (PNA), locked nucleic acid (LNA), morpholino        nucleotide, threose nucleic acid (TNA), glycol nucleic acid        (GNA), arabinose nucleic acid (ANA), 2′-fl uoroarabinose nucleic        acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol        nucleic acid (HNA), and/or unlocked nucleic acid (UNA); and/or        at least one nucleotide comprises a modified internucleoside        linker (e.g., wherein at least one phosphate of a nucleotide is        replaced by a modified internucleoside linker), wherein the        modified internucleoside linker is selected from        phosphorothioate, phosphorodithioate, phosphoramidate,        boranophosphonoate, an amide linker, and a compound of formula        (I)

where R³ is selected from O—, S—, NH₂, BH₃, CH₃, C₁₋₆ alkyl, C₆₋₁₀ aryl,C₁₋₆ alkoxy and C₆₋₁₀ aryl-oxy, wherein C₁₋₆ alkyl and C₆₋₁₀ aryl areunsubstituted or optionally independently substituted with 1 to 3 groupsindependently selected from halo, hydroxyl and NH₂; and R⁴ is selectedfrom O, S, NH, or CH₂.

-   -   23. The composition of embodiment 8, wherein the first two        base-pairing nucleotides on the 3′ end of the first and/or        second strand are modified.    -   24. The composition of embodiment 8, wherein the first two        base-pairing nucleotides on the 3′ end of the first and/or        second strand are 2′-MOE.    -   25. The composition of embodiment 8, wherein the 3′ terminal        phosphate of the first and/or second strands is replaced by a        modified internucleoside linker.    -   26. The composition of embodiment 8, wherein first and/or the        second strand is a sense strand comprising an 5′ end cap which        reduces the amount of the RNA interference mediated by the sense        strand.    -   27. In various embodiments, the sense strand comprises a 5′ end        cap selected: a nucleotide lacking a 5′ phosphate or 5′-OH; a        nucleotide lacking a 5′ phosphate or a 5′-OH and also comprising        a 2-OMe or 2′-MOE modification; 5′-deoxy-2′-O-methyl        modification; 5′-OME-dT; ddT; and 5′-OTr-dT.    -   28. A composition comprising a RNAi agent comprising a first        strand and a second strand, wherein the 3′-end of at least one        strand terminates in a phosphate or modified internucleoside        linker and further comprises a 3′ end cap, wherein the 3′ end        cap is selected from a compound of formula Ia or Ib or a        compound from any Table herein, wherein X is the first or second        strand, or any 3′ end cap disclosed herein; and wherein: (a) the        first and/or second strand is a 49-mer or shorter, is about 30        nucleotides long or shorter, is 19 nucleotides long, or between        15 and 49 nucleotides long; (b) optionally the RNAi agent has 1        or 2 blunt-ends or the RNAi agent comprises an overhang,        optionally a 1 to 6 nucleotide overhang on at least one 5′ end        or 3′ end; (c) optionally one or both strands are RNA or        optionally at least one nucleotide of the RNAi agent is        modified, wherein optionally said at least one modified        nucleotide is selected from among 2′ alkoxyribonucleotide, 2′        alkoxyalkoxy ribonucleotide, or 2′-fluoro ribonucleotide, and        optionally said at least one modified nucleotide is selected        from 2′-OMe, 2′-MOE and 2′-H; and wherein optionally the first        two base-pairing nucleotides on the 3′ end of the first and/or        second strand are modified, and optionally the first two        base-pairing nucleotides on the 3′ end of the first and/or        second strand are 2′-MOE; and wherein optionally one or more        nucleotides is modified or is DNA or is replaced by a peptide        nucleic acid (PNA), locked nucleic acid (LNA), morpholino        nucleotide, threose nucleic acid (TNA), glycol nucleic acid        (GNA), arabinose nucleic acid (ANA), 2′-fl uoroarabinose nucleic        acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol        nucleic acid (HNA), and/or unlocked nucleic acid (UNA); (d) at        least one nucleotide comprises a modified internucleoside        linker, wherein the modified internucleoside linker is selected        from phosphorothioate, phosphorodithioate, phosphoramidate,        boranophosphonoate, an amide linker, and a compound of formula        (I); and wherein optionally the 3′ terminal phosphate of the        first and/or second strands is replaced by a modified        internucleoside linker; and/or (e) optionally the first or the        second strand is a sense strand comprising an 5′ end cap which        reduces the amount of the RNA interference mediated by the sense        strand, wherein optionally the 5′ end cap selected a nucleotide        lacking a 5′ phosphate or 5′-OH; a nucleotide lacking a 5′        phosphate or a 5′-OH and also comprising a 2-OMe or 2′-MOE        modification; 5′-deoxy-2′-O-methyl modification; 5′-OME-dT; ddT;        and 5′-OTr-dT.    -   29. A composition comprising a RNAi agent comprising a first        strand and a second strand, wherein the 3′-end of at least one        strand terminates in a phosphate or modified internucleoside        linker and further comprises, in 5′ to 3′ order: a spacer, a        second phosphate or modified internucleoside linker, and a 3′        end cap, wherein the 3′ end cap is any 3′ end cap disclosed        herein or is selected from a compound of formula Ia or Ib or a        compound from any Table herein, wherein X is the first or second        strand which terminates in a phosphate or modified        internucleoside linker and further comprises, in 5′ to 3′ order:        a spacer, a second phosphate or modified internucleoside linker;        and wherein: (a) the first and/or second strand is a 49-mer or        shorter, is about 30 nucleotides long or shorter, is 19        nucleotides long, or between 15 and 49 nucleotides long; (b)        optionally the RNAi agent has 1 or 2 blunt-ends or the RNAi        agent comprises an overhang, optionally a 1 to 6 nucleotide        overhang on at least one 5′ end or 3′ end; (c) optionally one or        both strands are RNA or optionally at least one nucleotide of        the RNAi agent is modified, wherein optionally said at least one        modified nucleotide is selected from among 2′        alkoxyribonucleotide, 2′ alkoxyalkoxy ribonucleotide, or        2′-fluoro ribonucleotide, and optionally said at least one        modified nucleotide is selected from 2′-OMe, 2′-MOE and 2′-H;        and wherein optionally the first two base-pairing nucleotides on        the 3′ end of the first and/or second strand are modified, and        optionally the first two base-pairing nucleotides on the 3′ end        of the first and/or second strand are 2′-MOE; and wherein        optionally one or more nucleotides is modified or is DNA or is        replaced by a peptide nucleic acid (PNA), locked nucleic acid        (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol        nucleic acid (GNA), arabinose nucleic acid (ANA), 2′-fl        uoroarabinose nucleic acid (FANA), cyclohexene nucleic acid        (CeNA), anhydrohexitol nucleic acid (HNA), and/or unlocked        nucleic acid (UNA); (d) the spacer is a ribitol,        2′-deoxy-ribitol, diribitol, 2′-methoxyethoxy-ribitol (ribitol        with 2′-MOE), C3, C4, C5, C6, or 4-methoxybutane-1,3-diol; (e)        at least one nucleotide comprises a modified internucleoside        linker, wherein the modified internucleoside linker is selected        from phosphorothioate, phosphorodithioate, phosphoramidate,        boranophosphonoate, an amide linker, and a compound of formula        (I); and wherein optionally the 3′ terminal phosphate of the        first and/or second strands is replaced by a modified        internucleoside linker; and/or (f) optionally the first or the        second strand is a sense strand comprising an 5′ end cap which        reduces the amount of the RNA interference mediated by the sense        strand, wherein optionally the 5′ end cap selected a nucleotide        lacking a 5′ phosphate or 5′-OH; a nucleotide lacking a 5′        phosphate or a 5′-OH and also comprising a 2-OMe or 2′-MOE        modification; 5′-deoxy-2′-O-methyl modification; 5′-OME-dT; ddT;        and 5′-OTr-dT.    -   30. A composition comprising an RNAi agent of embodiment 25 and        a pharmaceutically acceptable carrier.    -   31. A composition comprising an RNAi agent of embodiment 25 and        a pharmaceutically acceptable carrier, for use as a medicament.    -   32. A method for inhibiting or reducing the level and/or        activity of a target gene in a cell comprising the step of        introducing into the cell one or more RNAi agent of embodiment        25.

Unless defined otherwise, the technical and scientific terms used hereinhave the same meaning as that usually understood by a specialistfamiliar with the field to which the disclosure belongs.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein. Unless indicated otherwise,each of the references cited herein is incorporated in its entirety byreference.

Claims are non-limiting and are provided below.

Although particular embodiments and claims have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, or is not intended to be limiting with respect to thescope of the appended claims, or the scope of subject matter of claimsof any corresponding future application. In particular, it iscontemplated by the inventors that various substitutions, alterations,and modifications may be made to the disclosure without departing fromthe spirit and scope of the disclosure as defined by the claims. Thechoice of nucleic acid starting material, clone of interest, or librarytype is believed to be a matter of routine for a person of ordinaryskill in the art with knowledge of the embodiments described herein.Other embodiments, advantages, and modifications considered to be withinthe scope of the following claims. Those skilled in the art willrecognize or be able to ascertain, using no more than routineexperimentation, many equivalents of the specific embodiments of thedisclosure described herein. Such equivalents are intended to beencompassed by the following claims. Redrafting of claim scope in laterfiled corresponding applications may be due to limitations by the patentlaws of various countries and should not be interpreted as giving upsubject matter of the claims.

1. A compound of formula Ia:

in which: X is H; OH, wherein the hydroxyl group can optionally befunctionalized as succinate or attached to a solid support; ODMT;carboxylic acid; the 3′ end of a strand of a RNAi agent; or the 3′ endof a molecule comprising a strand of a RNAi agent, wherein the 3′ end ofthe strand terminates in a phosphate or modified internucleoside linkerand further comprises in 5′ to 3′ order: a spacer, and a secondphosphate or modified internucleoside linker; Y is CH or N; m is 0 or 1;p is 1, 2 or 3; R₃ is hydrogen, 2-(hydroxy-methyl)-benzyl,3-(hydroxy-methyl)-benzyl, succinate, or a solid support; wherein the(CH₂)_(m)—O—R₃ moiety is attached to the phenyl ring at position 3 or 4;R₄ is hydrogen; R₅ is hydrogen; or R₄ and R₅, together with the phenylrings to which R₄ and R₅ are attached, form 6H-benzo[c]chromene. 2-32.(canceled)
 33. A compound of formula Ib:

wherein: X is H; OH, wherein the hydroxyl group can optionally befunctionalized as succinate or attached to a solid support; ODMT; or acarboxylic acid; q is 0, 1 or 2; R₆ is phenyl which is unsubstituted orsubstituted with a group selected from benzoxy and 3,4-dihydroxybutyl;R₇ is hydrogen or hydroxy-ethyl, wherein if R₇ is hydroxy-ethyl, thehydroxyl is optionally functionalized as succinate or attached to asolid support; R₈ is hydrogen or methoxy; Y₁ is CH or N; and Y₂ is N orCR₉; wherein R₉ is selected from hydrogen and methyl.
 34. The compoundof claim 33 selected from:


35. An RNAi agent of formula Ib:

wherein: X comprises a strand of the RNAi agent; q is 0, 1 or 2; R₆ isphenyl which is unsubstituted or substituted with a group selected frombenzoxy and 3,4-dihydroxybutyl; R₇ is hydrogen or hydroxy-ethyl, whereinif R₇ is hydroxy-ethyl, the hydroxyl is optionally functionalized assuccinate or attached to a solid support; R₈ is hydrogen or methoxy; Y₁is CH or N; and Y₂ is N or CR₉; wherein R₉ is selected from hydrogen andmethyl.
 36. The RNAi agent of claim 35, wherein X is attached at the 3′end of a strand of the RNAi agent.
 37. The RNAi agent of claim 36,wherein the strand of the RNAi agent terminates at its 3′ end in aphosphate or modified internucleoside linker, and X is attached at thephosphate or modified internucleoside linker.
 38. The RNAi agent ofclaim 36, wherein the strand of the RNAi agent terminates at its 3′ endin a phosphate or modified internucleoside linker, the phosphate ormodified internucleoside linker is substituted by a spacer, which issubstituted by a second phosphate or modified internucleoside linker, in5′ to 3′ order, and X is attached at the second phosphate or modifiedinternucleoside linker.
 39. The RNAi agent of claim 35 selected from:


40. The RNAi agent of claim 35, wherein the strand of the RNAi agent isa first strand and X further comprises a second strand.
 41. The RNAiagent of claim 40, wherein the first strand is an antisense strand. 42.The RNAi agent of claim 40, wherein the first and/or second strands areno more than about 30 nucleotides long.
 43. The RNAi agent of claim 40having 1 or 2 blunt ends.
 44. The RNAi agent of claim 40 having anoverhang on at least one 5′ end or 3′ end.
 45. The RNAi agent of claim40, wherein X further comprises a spacer.
 46. The RNAi agent of claim45, wherein the spacer is an abasic nucleotide.
 47. The RNAi agent ofclaim 45, wherein the spacer is a ribitol, 2′-deoxy-ribitol, diribitol,2′-methoxyethoxy-ribitol (ribitol with 2′-MOE), C3, C4, C5, C6, or4-methoxybutane-1,3-diol.
 48. The RNAi agent of claim 40, wherein atleast one nucleotide of the RNAi agent is modified.
 49. The RNAi agentof claim 48, wherein the at least one modified nucleotide is selectedfrom 2′ alkoxyribonucleotide, 2′ alkoxyalkoxy ribonucleotide, 2′-fluororibonucleotide, 2′-OMe, 2′-MOE and 2′-H.
 50. The RNAi agent of claim 40,wherein one or more nucleotides is modified, is DNA, or is replaced by apeptide nucleic acid (PNA), locked nucleic acid (LNA), morpholinonucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA),arabinose nucleic acid (ANA), 2′-fluoroarabinose nucleic acid (FANA),cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA),and/or unlocked nucleic acid (UNA); and/or at least one nucleotidecomprises a modified internucleoside linker, wherein the modifiedinternucleoside linker is selected from phosphorothioate,phosphorodithioate, phosphoramidate, boranophosphonoate, an amidelinker, and a compound of formula (I)

where R³ is selected from O⁻, S⁻, NH₂, BH₃, CH₃, C₁₋₆ alkyl, C₆₋₁₀ aryl,C₁₋₆ alkoxy and C₆₋₁₀ aryl-oxy, wherein C₁₋₆ alkyl and C₆₋₁₀ aryl areunsubstituted or optionally independently substituted with 1 to 3 groupsindependently selected from halo, hydroxyl and NH₂; and R⁴ is selectedfrom O, S, NH, or CH₂.
 51. The RNAi agent of claim 40 comprising a sensestrand, the sense strand comprising a 5′ end cap selected from anucleotide lacking a 5′ phosphate or 5′-OH; a nucleotide lacking a 5′phosphate or a 5′-OH and also comprising a 2-OMe or 2′-MOE modification;5′-deoxy-2′-O-methyl modification; 5′-OME-dT; ddT; and 5′-OTr-dT.
 52. Acomposition comprising the RNAi agent of claim 35 and a pharmaceuticallyacceptable carrier.
 53. A method for inhibiting or reducing the leveland/or activity of a target gene in a cell comprising the step ofintroducing into the cell the RNAi agent of claim
 40. 54. A method ofpreparing the RNAi agent of claim 35 comprising: reacting a strand ofthe RNAi agent with a compound of formula

wherein: X is OH, wherein the hydroxyl group can optionally befunctionalized as succinate or attached to a solid support; ODMT; or acarboxylic acid; q is 0, 1 or 2; R₆ is phenyl which is unsubstituted orsubstituted with a group selected from benzoxy and 3,4-dihydroxybutyl;R₇ is hydrogen or hydroxy-ethyl, wherein if R₇ is hydroxy-ethyl, thehydroxyl is optionally functionalized as succinate or attached to asolid support; R₈ is hydrogen or methoxy; Y₁ is CH or N; and Y₂ is N orCR₉; wherein R₉ is selected from hydrogen and methyl.